A Gene Encoding for an Amylase From Thermophilic Bacillus Sp Strain TS-23

Journal of Applied Microbiology 1997, 82, 325–334

A gene encoding for an a-amylase from thermophilic Bacillussp. strain TS-23 and its expression in Escherichia coli

L.-L. Lin, W.-H. Hsu 1 and W.-S.Chu 2

Department of Food Nutrition, Hung Kuang Institute of Medical and Nursing Technology, Shalu, Taichung, 1Instituteof Molecular Biology, National Chung Hsing University, Taichung, and 2Culture Collection and Research Center, FoodIndustry Research and Development Institute, Hsinchu, Taiwan

5561/11/95: received 21 November 1995, revised 6 August 1996 and accepted 7 August 1996

L.-L. LIN, W.-H. HSU AND W.-S. CHU. 1997. An a-amylase gene from Bacillus sp. strain TS-23was cloned and expressed by using its own promoter on the recombinant plasmid pTS917 inEscherichia coli. A cell fractionation experiment revealed that approximately 60% of theamylase activity was in the periplasmic space. Analysis and activity staining of theconcentrated supernatant fraction by SDS-polyacrylamide gel electrophoresis showed anapparent 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 acalculated mol. wt of 69 543. The predicted amino acid sequence showed high homologywith Bacillus species, E. coli and Salmonella typhimurium a-amylases. Deletion of 96 aminoacids from the C-terminal portion of the amylase did not result in the loss of amylolyticactivity. 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.

INTRODUCTION homology in endoamylases. For instance, B. licheniformis, B.megaterium, B. amyloliquefaciens and B. subtilis are generally

Starch, as the primary storage polysaccharide in plants, isgrouped together (Priest et al. 1988), but only the B. amy-

degraded by amylolytic enzymes from numerous micro-loliquefaciens and B. licheniformis a-amylases are similar which

organisms (Vihinen and Mantsala 1989). Starch-degradingform a group with those from B. stearothermophilus and Bacil-

enzymes can be grouped into endo- and exoamylase by theirlus sp. strain 707 (Tsukamoto et al. 1988).

types of action towards substrates. The endoamylases canRecently, the general features of extracellular endoamy-

cleave internal a-1,4 and/or a-1,6 linkages in starch whilelases from the thermophilic Bacillus sp. strain TS-23 have

the exoamylases split the a-1,4 bonds of a-glucan from thebeen characterized (Lin et al. 1994). In this paper, we report

non-reducing ends. It has been reported that nearly all thethe cloning, sequencing and expression of a gene coding for

endoamylases sequenced to date have homologous regionsan amylase from strain TS-23. The deduced amino acid

(Mackay et al. 1985 ; Rogers 1985 ; Nakajima et al. 1986 ;sequence of the cloned polypeptide (AmyA) was also com-

Svensson 1988 ; MacGregor and Svensson 1989 ; Svenssonpared with sequences for amylases of prokaryotic origin.

et al. 1989). In addition, an intracellular glucosidase of Strep-tococcus mutans (Russell and Ferretti 1990) and a branchingenzyme from Escherichia coli (Baecker et al. 1986) also contain

MATERIALS AND METHODSthe homologous sequences to endoamylases. Numeroussequence comparisons reveal four generally recognized areas

Bacterials strains, plasmids and growth conditionsof homology (designated regions I, II, III and IV) among theendoamylases and there also appears to be five additional The bacteria used included Bacillus sp. strain TS-23 (Linregions of high similarity (Rumbak et al. 1991). However, et al. 1994), Escherichia coli DH5a (supE44 DlacU169 (f80closely related Bacillus species do not necessarily have high lacZDM15) hadR17 recA1 endA1 gyrA96 thi-1 relA1 ; Han-

ahan 1983), E. coli JM101 (supE thiD[lac-proAB]F?[traD36Correspondence to : Dr Long-Liu Lin, Department of Food Nutrition, HungproAB¦ lacIq lacZDM15] ; Yanisch-Perron et al. 1985), E.Kuang Institute of Medical and Nursing Technology, 34 Chungchie Road, Shalu,

Taichung, Taiwan. coli NovaBlue (endA1 hsdR17 [rk12−mk12¦] supE44 thi-1

© 1997 The Society for Applied Bacteriology

326 L.-L. LIN ET AL.

gyrA96 relA1 lac[F? proA¦B¦ lacIqZDM15 ::Tn10(tetR)] ; dard assay mixtures (total volume 1 ml) contained 250 ml of1% soluble starch, 250 ml of NaOH–glycine buffer (50 mmolNovagen Inc., Madison, WI, USA) and the K-12-derived E.

coli strain M15(pREP4) (Nals Strs rifs lac− ara− gal− F− l−1, pH 8·8) and 500 ml of appropriate amount of enzyme.After 10 min incubation at 70°C, the reaction was stoppedrecA¦ uvr¦ ; Qiagen GmbH, Hilden, Germany). The plas-

mids used were pUC119 (Vieira and Messing 1987) and the by addition of 1 ml of dinitrosalicylic acid solution (Bern-feld 1955), heated at 100°C for 5 min and the absorbanceQiagen Type IV expression vectors (Qiagen). The medium

and growth conditions for Bacillus sp. strain TS-23 were at 575 nm was determined. One unit of amylase liberates1 mmol l−1 of reducing groups (as glucose equivalents) perdescribed previously (Lin et al. 1994). Escherichia coli strains

were grown in Luria–Bertani (LB) medium (Sambrook et al. min. As controls for E. coli periplasmic and intracellularenzymes, alkaline phosphatase and malate dehydrogenase1989).activities were assayed by the methods of Brickman andBeckwith (1975) and Kitto (1969), respectively. Protein con-

DNA manipulationscentrations were determined by the method of Bradford(1976) with bovine albumin as a standard.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 (Ish-

Electrophoresis and detection of amylase activity inHorowicz and Burke 1981). One-step preparation of com-

gelspetent cells and plasmid transformations were performed bythe method of Chung et al. (1989). The digestion of DNA Sodium dodecyl sulphate–polyacrylamide gel electrophoresis

(SDS-PAGE) was done with a slab gel as described bywith restriction endonucleases was carried out according tothe manufacturer’s directions. Restriction mapping and other Laemmli (1970). Proteins were stained with Coomassie bril-

liant blue R-250 (Weber and Osborn 1969). The markerroutine molecular methods used in this work were describedby Sambrook et al. (1989). 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), car-

Construction and screening of the genomic librarybonic anhydrase (31 000) and trypsin inhibitor (21 500).

Amylase activity gels were performed on 10% SDS-PAGEChromosomal DNA of Bacillus sp. strain TS-23 was partiallydigested with Sau3AI and size fractionated in a 1% agarose with concentrated supernatant fractions of E. coli JM101

cultures containing pUC119 and pTS917. The amylolyticgel. The DNA fragments with size between 3 and 8 kb wasrecovered by the Geneclean II® kit (Bio 101 Inc., La Jolla, activity band was detected in situ after electrophoresis and

renaturation of proteins according to Lacks and SpringhornCA, USA) and ligated into BamHI-cleaved pUC119 with T4DNA ligase after treatment by alkaline phosphatase. The (1980).ligation mixture was then introduced into E. coli DH5a bytransformation. The transformants were selected on LB plate

N-terminal amino acid analysiscontaining 100 mg ml−1 ampicillin and 0·5% starch azure(Sigma Chemical Co., St Louis, MO, USA). Transformants The concentrated supernatant proteins from E. coli JM101

(pTS917) were separated by 10% SDS-PAGE gels. Proteinsshowing amylase activity were identified by a clear zonearound the colony. were electrotransferred in 10 mmol l−1 3-(cyclohexylamino)-

1-propanesulphonic acid (CAPS)–10% methanol (pH 11·0)to an Immobilon-P-polyvinylidene difluoride membrane

Localization of amylase(Millipore, Bedford, MA, USA) at 50 V for 30 min andvisualized by staining with 0·1% Coomassie brilliant blue R-Exponentially-growing cells were washed twice with 50 mmol

l−1 NaOH–glycine buffer (pH 8·8), and subjected to osmotic 250 in 50% methanol as described previously (Matsudaira1987). The desired band was cut out of the membrane, driedshock as described by Nossal and Heppel (1966). The shocked

cells were disrupted by passage through a French Press (SLM and sequenced in an 477A protein sequence (Applied Bio-system).Instrument Inc., Urbana, IL, USA). The resulting fractions

were assayed for the activities of amylase, malate dehydro-genase and alkaline phosphatase.

Nucleotide sequencing and data analysis

The templates for sequencing were obtained by constructingEnzyme assays

a set of exonuclease III-generated deletions from SacI or PstIsite, which closely presents at either end of the insert, ofThe amylase activity was determined by measuring the enzy-

matic release of reducing groups from soluble starch. Stan- pTS246 as described by Henikoff (1984) and detailed in the

© 1997 The Society for Applied Bacteriology, Journal of Applied Microbiology 82, 325–334

AN AMYLASE GENE OF BACILLUS SP. TS-23 327

Erase-a-Base Kit (Promega, Madison, WI, USA) protocol. (Qiagen). The resulting material was diluted 1 : 100 in refold-ing buffer (50 mmol l−1 NaOH–glycine buffer, pH 8·8/100The deleted plasmids were sequenced with the Taq Dye

Terminator Cycle Sequencing Kit (Applied Biosystems) mmol l−1 NaCl/1 mmol l−1 EDTA/1 mmol l−1 reduced and1·2 mmol l−1 oxidized glutathione). The refolding reactionusing M13 forward and reverse primers. Gaps in the sequence

were determined by the use of appropriate primers and the proceeded for 12 h at 4°C with stirring. The renatured proteinwas dialysed against 2 l of refolding buffer at 4°C for 16 hDye Terminator Kit. DNA sequence data were obtained from

the ABI model 473A Automated Sequencer and the program with one buffer change and concentrated by ultrafiltration onAmicon concentration apparatus (Amicon, Lexington, MA,SEQED (ABI) was used to edit the data. Unless otherwise

indicated, both strands of the DNA were sequenced. The USA).SEQMAN and GENEMAN (DNASTAR Inc., Madison,WI, USA) software packages were used for analysis of DNA

Nucleotide sequence accession numberand protein sequences. The program FASTP was used tosearch database for protein sequences similar to that predicted The nucleotide sequence of amyA and the flanking regions

reported here have been deposited in the Genbank databasefrom the DNA sequence. Multiple alignment of proteinsequences was achieved with the program CLUSTAL. under the accession number U22045.

RESULTSConstruction and overexpression of amyA deletionderivatives

Isolation and location of amylase geneThe synthetic primers (forward primers : 5?-GAAC-GATGGATCCCTTTGGA-3? and 5?-GCCGCAGGGAT- From approximately 8000 colonies, three E. coli DH5a trans-

formants that produced a distinct halo around the coloniesCCAAGTATA-3? ; reverse primer : 5?-TGGCACACTGC-AGCTCGCCG-3?) and Qiagen Type IV expression vectors on starch azure plates were detected. Similar halos were

produced by the colonies when they were plated onto starchwere used for the polymerase chain reaction (PCR)-aidedconstruction of plasmids encoding truncated AmyA plates stained with I2–KI solution (0·01 mol l−1 −0·1 mol

l−1 KI). Restriction enzyme analysis showed that the plasmidsderivatives. Amplification of DNA fragments via the PCRwas performed by a GeneAmp PCR system 9600 (Perkin from three clones had different-sized overlapping inserts and

the plasmid, pTS917, with the largest insert was chosen forElmer Cetus, Norwalk, CT, USA). Reaction mixtures (100ml) contained 1·25 mmol l−1 of the four dNTPs, 20 mmol l−1 further study. A restriction map of the 3·18 kb insert fragment

on pTS917 was constructed (Fig. 1). The 2·92 kb XbaI–XbaIof each primer, 100 ng of template DNA, 1·5 mmol l−1

of MgCl2, 10 mmol l−1 of Tris–HCl (pH 8·3) and 2·5 units fragment was subcloned into pUC119 in both orientations.Both subclones (pTS245 and pTS246) retained the ability toof Taq DNA polymerase (Stratagene, La Jolla, CA, USA).

The amplification cycle was : denaturation of the DNA at produce halos on starch plates, suggesting that an endogenouspromoter was present on the insert. Exonuclease III deletions94°C for 2 min, annealing at 55°C for 1·5 min and primer

extension at 72°C for 2 min. Up to 25 cycles of amplification in pTS246 from SacI site (pTS301) or PstI site (pTS401,pTS402 and pTS403) retained amylolytic activity, while thewere employed. The BamHI and PstI sites were used for

insertion of the PCR products into the corresponding sites further deleted clones (pTS302 or pTS404) lost enzymeactivity (Fig. 1).of expression vectors, thus yielding the plasmids pEX1 and

pEX2, respectively. The resulting plasmids contain 6×Histag fused in frame with the AmyADN50 (pEX1) and

Localization of amylolytic activity in E. coliAmyADN126 (pEX2). The hybrid genes are under the tran-scriptional control of the phage T5 promoter, which is regu- The localization of enzymatic activity in E. coli cells was

determined by using the full-length plasmid pTS917 andlated via the product of the lacI gene (Farabaugh 1978).Escherichia coli M15 (pREP4) cells harbouring pEX1 or the shortened plasmid pTS403. As a control, E. coli JM101

harbouring pUC119 was cultivated under the samepEX2 were grown in 500 ml of LB containing 100 mg ml−1

ampicillin and 25 mg ml−1 kanamycin to an O.D.600 of about conditions. The result showed that no significant amylaseactivity was detected in any fractions of E. coli0·7. IPTG was added to a final concentration of 2 mmol l−1

and the incubation was continued at 37°C for 5 h. The cells JM101(pUC119) while the enzyme activity in the host cellsharbouring pTS917 or pTS403 was very similar, with thewere harvested by centrifugation at 4000 g for 20 min. For

preparation of freeze-thaw crude extracts, the cells were periplasmic fraction containing approximately 60% of theamylolytic activity (Table 1). It is interesting to note thatstored at −80°C for 2 h and thawed at room temperature for

15 min. The recovery of gene products under denaturing about 25% of the total alkaline phosphatase and 35% of totalamylase activity were found in the supernatant fractions ofconditions was performed as recommended by the supplier



pTS246

pTS301pTS302pTS401pTS402pTS403pTS404

pTS917

+

+–+++–

Amylaseactivity

+1 kb

amyA

X E N K A XPlac

Fig. 1 Restriction enzyme map and deletion analysis of DNAinsert of pTS917. The open circle and arrow denote theposition and direction of the lacZ promoter. The approximateopen reading frame and the direction of transcription are Fig. 2 SDS-PAGE (A) and activity staining (B) analysis ofindicated by a solid arrow. The cleavage sites are for XbaI (X), extracellular proteins of Escherichia coli cells carrying pTS917 andEcoRV (E), NdeI (N), KpnI (K) and AvaII (A). Amylase pUC119. Lanes : M, Mol. wt standards ; 1, supernatant fractionactivity was measured by the hydrolysis of soluble starch in LB of E. coli (pUC119) ; 2, supernatant fraction of E. coli (pTS917)agar plates

E. coli JM101 cells containing pTS917 or pTS403 (Table 1). 69 543 (Fig. 3). The amylase-encoding sequence starts withan ATG start codon preceded by 5 bp of spacing by a potentialSDS-PAGE analysis and activity staining of the proteins in

the culture broth from E. coli JM101 (pTS917) cells showed ribosome-binding sequence (5?-AAGGATGTG-3?) withcomplementarity to the 3? end of the E. coli 16S rRNAa protein band with an apparent mol. wt of approximately

65 000 (Fig. 2). (3?-AUUCCUCCACUAG-5?) (Shine and Dalgarno 1974 ;Stormo et al. 1982). A putative promoter region (TTGTAA-N17-TATAAT) with strong homology to the E. coli (s70)

Sequence analysisconsensus promoter (Hawley and McClure 1983) was located69 bp upstream of the ATG start codon. The translationalThe nucleotide sequence of the 2447-bp fragment from

pTS246 revealed a single open reading frame (ORF) encoding stop codon TAA is followed by a stem-loop structure Thisstructure may serve as a rho-independent terminator in E.a protein of 613 amino acids with a calculated mol. wt of

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 MalatePlasmid Fraction Amylase phosphatase dehydrogenase—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––pUC119 Supernatant ND 5–10 ND

Periplasmic ND 78–89 NDCytoplasmic ND 6–12 100

pTS917 Supernatant 28–37 21–29 0–3Periplasmic 51–62 66–70 2–7Cytoplasmic 10–18 5–9 93–95

pTS403 Supernatant 21–35 25–31 3–6Periplasmic 46–63 50–68 0–5Cytoplasmic 16–19 7–19 92–94

—––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––––

ND, No enzyme activity was detected.



Fig. 3 Nucleotide sequence of the Bacillus sp. strain TS-23 amyA structural gene and its flanking regions. The predicted aminoacid sequence of the AmyA is given below in single-letter code. Consensus −10 and −35 regions of amyA promoter are underlined andthe ribosome-binding site (RBS) is boxed. Inverted repeat sequences are shown by converging arrows. A downward arrow indicatesthe putative cleavage site of the signal peptide. The amino acids identical to those from N-terminal sequence are circled

coli. The G¦C content of the Bacillus sp. strain TS-23 amyA but when 180 bp (positions 1 to 180) were removed, theresulting clone (pTS302) did not have amylase activity (Figsgene was 39·7% and the upstream regulatory region was

38%. 1 and 3). This indicates that the upstream region must playa role in the expression of amyA gene.The AmyA C-terminus deletions synthesized by plasmids

pTS401, pTS402 and pTS403 showed no apparent change The N-terminal sequence of the secreted amylase wasdetermined as Asn–Thr–Ala–Pro–Ile, which indicates thein amylolytic activity, indicating the last 96 amino acids could

be removed without loss of activity. However, removing the initial 30 amino acids (Met-1 to Ala-30) were cleaved duringexport of the protein. Indeed, the properties of the N-ter-last 189 amino acids (pTS404) abolished amylase activity

(Fig. 1). Deletion of 14 bp (positions 1 to 14 ; pTS301) from minal region in the immature amylase are in good agreementwith the requirements that have been proposed for exportthe upstream region did not affect the expression of amyA,



signal sequences (von Heijne 1983 ; Gierasch 1989). This wt of about 57 000 was also produced in IPTG-induced cellsof E. coli M15 (pREP4) harbouring pEX2 (Fig. 5). Aftersignal contains a cluster of positively charged amino acids

adjacent to the fMet residue at the N-terminus, followed by denaturation and refolding of the induced protein, about 0·8mg of AmyAD126 per l of growth medium was obtaineda region rich in hydrophobic amino acids (Fig. 3). There are

two Ala residues at position −3 and −1 with respect to the while the recovered protein showed no amylolytic activity.most likely cleavage site calculated according to the rules ofvon Heijne (1986).

DISCUSSION

A gene (amyA) encoding amylase of Bacillus sp. strain TS-Comparison with other amylases

23, a thermophilic and alkaliphilic bacterium isolated from ahot spring in Taiwan, was successfully cloned and expressedThe predicted amino acid sequence of the amylase was com-

pared with sequences in the Swiss-Prot data base (Translated in E. coli. The amyA gene codes for a polypeptide with anapparent mol. wt of 69 543. The size of this polypeptide isRelease 85) by using the FASTP program. Homology was

found between the AmyA enzyme and the a-amylase of B. similar to typical microbial a-amylases (Vihinen and Mantsala1989). Other large amylases, such as the amylase of Bacillusstearothermophilus (Ihara et al. 1985) as well as the amylases

of Bacillus sp. strain 707 (Tsukamoto et al. 1988), B. amy- polymyxa (Uozumi et al. 1989), the G6-amylase of Bacillussp. strain H-167 (Shirokizawa et al. 1990) and the G4-amylaseloliquefaciens (Takkinen et al. 1983), B. licheniformis (Yuuki

et al. 1985), B. circulans (EMBL accession no. X60779), Sal- of a Micrococcus sp. (Kimura and Horikoshi 1990), usuallyhave additional properties. The amylase of B. polymyxa hasmonella typhimurium (Raha et al. 1991) and E. coli (Raha et

al. 1991). In addition to regions corresponding to the four both a- and b-amylolytic activity while Bacillus sp. strain H-167 and the Micrococcus sp. amylases have a specific exoamy-highly conserved domains (regions I, II, III, IV) in endoamy-

lases (Rogers 1985 ; Nakajima et al. 1986 ; Svensson 1988), lolytic activity associated with them. It has been reported thatthe C-terminal region of Butyrivibrio fibrisolvens amylase wasthere are a number of other conserved regions and residues

present in these amylases (Fig. 4). The degree of amino acid not essential for amylolytic activity (Rumbak et al. 1991).Similarly, deletion of approximately 16% of the C-terminalidentity between AmyA and a-amylase of B. ste-

arothermophilus was 77% and between AmyA and the Bacillus portion of the Bacillus sp. strain TS-23 amylase did not resultin loss of enzymatic activity. However, the biological functionsp. 707 maltohexaose-producing amylase was 64% (Table 2).

When amino acid replacement by conserved amino acids was of this region is unclear.The leader sequences of Bacillus spp. vary between 18 andtaken into account, the similarity increased to 80% and 68%,

respectively. As shown in Table 2, the alignment of AmyA 35 amino acid residues in length (Simonen and Palva 1993).Likewise, the first 30 amino acids of Bacillus sp. strain TS-with five other amylases showed identity between 39 and

61%. 23 amylase have structural features of single peptide (vonHeijne 1983). This signal peptide was functional in E. colisince most of the amylase protein was exported to the

Production and purification of AmyA deletionperiplasm. It was of interest to note that about 35% of the

derivativesamylolytic activity was present in the culture broth andapproximately 25% of alkaline phosphatase activity, the peri-The deletions from the 5? end of the ORF of amyA gene

were linked to a fusion sequence including the codes of six plasm control enzyme, was also detected in the extracellularfluid. In contrast, the malate dehydrogenase was pre-consecutive histidine residues. Production of AmyA deletion

derivatives was evaluated by inducing the shaker flask cultures dominantly detected in the cytoplasmic portion, indicatingleakage of cytoplasmic proteins did not occur (Table 1). Theseof E. coli M15 (pREP4) harbouring pEX1 and pEX2, respec-

tively. Synthesis of an approximately 62-kDa protein was results suggest that a-amylase of strain TS-23 readily trans-locates across the cytoplasmic membrane and subsequentlydetected in IPTG-induced cells containing pEX1 and was

barely observable in uninduced sample (Fig. 5). Densi- leaks to the culture broth. A similar result has been foundin B. stearothermophilus a-amylase overexpressed in E. colitometric gel scanning indicated that the band comprised

about 28% of total cellular proteins. Purification of (Suominen et al. 1987).Evidence from nuclear resonance studies of the porcineAmyADN50 from E. coli involved affinity chromatography

with nickel-agarose which made use of the stretch of his- pancreatic a-amylase suggests that catalysis occurs by a nucle-ophilic attack on C-1 of the incipient reducing sugar (Tao ettidines at the N-terminus of AmyADN50. The refolding

procedure resulted in a homogeneous protein, as judged by al. 1989). This reaction would require an electrophile todonate a proton to the sugar-leaving group. On the basis ofSDS-PAGE (data not shown), with a specific activity of 250

U per mg of protein and a yield of 10·3 mg of AmyAD50 per homology and structure, this electrophile has been proposedto be a carboxyl group (Matsuura et al. 1984 ; Buisson et al.l of growth medium. A protein band with an apparent mol.



Fig. 4 Alignment of predicted amino acid sequence with prokaryotic amylase. Symbols denote : Amy2-Salty, Salmonellatyphimurium ; 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 agreementof the amino acid in at least three sequences with that determined for the consensus strand. The previously recognizedregions I to IV are boxed. Homology was maximized by introducing gaps denoted by a dot



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 77·2 63·7 60·7 60·7 45·2 39·9 38·62. Bst 548 100 60·8 59·1 59·6 44·6 38·7 37·23. Bac2 518 100 61·9 63·1 48·3 39·9 39·44. Bam 514 100 76·2 45·2 38·7 37·85. Bli 512 100 45·6 39·5 38·46. Bci 493 100 41·2 41·27. Sty 494 100 87·48. 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.

binding residues suggests that these residues play importantroles in catalytic function and substrate binding. The align-ment of the amino acid sequence of Bacillus sp. strain TS-23a-amylase with seven other amylases not only showed fourregions highly conserved in all enzymes, but also revealedthe conservation of several additional regions. However, thefunctional significance of these regions is unclear.

High-level production of heterologous proteins in E. colioften accumulate intracellularly insoluble aggregates, termedinclusion bodies. Previously, considerable efforts have been

Fig. 5 SDS-PAGE analysis of the truncated amylase derivatives. made to express recombinant proteins in more soluble, activeThe proteins of cell-free extracts were separated by 10% form (Lunn et al. 1986 ; Goloubinoff et al. 1989 ; Takagi etSDS-PAGE and stained with Coomassie brilliant blue. Lanes : al. 1990). In our case, no amylase activity was detected in theM, Mol. wt standards ; 1 and 3, Escherichia coli cells cell-free extract of IPTG-induced cells, and the purificationcontaining pEX1 with (lane 3) or without (lane 1) IPTG of native proteins under non-denaturing conditions did notinduction ; 2 and 4, E. coli cells carrying pEX2 with (lane 4) or

succeed (data not shown), suggesting that the overexpressionwithout (lane 2) IPTG inductionof AmyADN50 in E. coli system resulted in the formation ofinclusion bodies. Although the advantages for production of

1987 ; Vihinen et al. 1990). Three aspartic acids (in regions soluble proteins have been documented (Shatzman 1990),I, II and IV, respectively) and one glutamic acid (region III) inclusion bodies are sometimes helpful in that they are readilyhave been suggested as catalytic residues. In strain TS-23 a- amenable to purification and they may also stabilize proteinsamylase, three residues, Asp-132, Asp-265 and Asp-362 (in that otherwise might be subject to degradation by host pro-regions I, II and IV, respectively), and one glutamic acid, teases (Cheng et al. 1981). Recently, Janknecht et al. (1991)Glu-295 (in region III), are conserved (Fig. 4). Amino acids have exploited a method for affinity purification of histidine-implicated in substrate binding have generally been localized tagged proteins. By this method, we have purified the recom-to high-similatory regions (Matsuura et al. 1984 ; Buisson et binant AmyADN50 with a yield of 10·3 mg l−1 of cultureal. 1987). These include the highly conserved residues His- broth. It is worth noting that the denatured protein could be106 (region I), Asp-229 (region II), His-238 (region II), Glu- refolded to yield an activable enzyme, indicating that the first264 (region II) and His-330 (region IV). Another conserved 50 amino acids of AmyA were not important for amylolyticamino acid (Arg-232 in region II) in B. stearothermophilus is activity.necessary for activity and may be needed to electrostatically In conclusion, we have cloned and sequenced an a-amylasehold substrate binding and/or active-site residues in the pro- gene from the alkaliphilic and thermophilic bacterium Bacil-per configuration (Vihinen et al. 1990). In fact, all these lus sp. strain TS-23. The cloned amylase has a signal peptideresidues were clearly present in Bacillus sp. strain TS-23 a- typical for proteins exported by Gram-positive bacteria and

about 35% of amylolytic activity was found extracellularly.amylase. The presence of putative active sites and substrate



Laboratory Manual. pp. 70–102. Norwich, UK : The John InnesThus, it would be of interest to study the effects of signalFoundation.peptide mutations on processing of this enzyme in E. coli.

Ihara, H., Sasaki, T., Tsuboi, A., Yamagata, H., Tsukago, N. andUdaka, S. (1985) Complete nucleotide sequence of a thermophilica-amylase : homology between prokaryotic and eukaryotic a-

ACKNOWLEDGEMENTS amylases at the active sites. Journal of Biochemistry 98, 95–103.Ish-Horowicz, D. and Burke, J.F. (1981) Rapid and efficient cosmidThe authors thank Dr C.P. Tseng (Institute of Biote-

cloning. Nucleic Acids Research 14, 8605–8613.chnology, National Chiao Tung University, Taiwan) for valu-Janknecht, R., Martynoff, G., Lou, J., Hipskind, R.A., Nordheim,able comments on the manuscript. This work was supported

A. and Stunnenberg, H.G. (1991) Rapid and efficient purificationin part by research grants from the Council of Agricultureof native histidine-tagged protein expressed by recombinant vac-

and Ministry of Economic Affairs of the Republic of China.cinia virus. Proceedings of the National Academy of Sciences USA88, 8972–8976.

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A Gene Encoding for an Amylase From Thermophilic Bacillus Sp Strain TS-23