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Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8
Research in Microbiology 154 (2003) 681–687 www.elsevier.com/locate/resmic
Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8 Woo Jin Lim a , Sang Ryeol Park a , Chang Long An a , Jong Yeoul Lee a , Su Young Hong a , Eun Chule Shin a , Eun Ju Kim a , Jong Ok Kim c , Hoon Kim d , Han Dae Yun a,b,∗ a Division of Applied Life Science, Gyeongsang National University, Chinju 660–701, Republic of Korea b Research Institute of Life Science, Gyeongsang National University, Chinju 660–701, Republic of Korea c Takara Korea Biomedical Inc. R & D Center, Uiwang 437–020, Republic of Korea d Department of Agricultural Chemistry, Sunchon National University, Sunchon 540–742, Republic of Korea
Received 10 February 2003; accepted 5 September 2003 First published online 9 September 2003
Abstract The gene encoding an intracellular α-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed α-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50 187 Da). The molecular weight of the enzyme was estimated to be 50 000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among α-amylases. The AmyB α-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 ◦ C. Like the α-amylases of many other organisms, the thermostability of T. maritima MSB8 α-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca2+ (10 mM). 2003 Elsevier SAS. All rights reserved. Keywords: Thermotoga maritima; Intracellular α-amylase; Starch-nondenaturing PAGE
1. Introduction Hyperthermophilic bacteria and archaea, which grow optimally above 80 ◦ C, are of considerable interest because of the hyperthermostability of their enzymes [19]. In industry, hyperthermophilic enzymes could be advantageous not only for their extreme thermostability but also because of their resistance to denaturing agents, solvents, and proteolytic enzymes [3]. The archaea Thermatoga maritima is capable of utilizing simple carbohydrates as well as complex polysaccharides as energy sources. These include glucose, sucrose, cellulose, xylan and starch, the carbohydrate of interest here [6]. Schumann et al. [18] reported the existence of hydrolytic starch-degrading activities in T. maritima, but the activity levels seemed too low to allow purification and characterization. α-Amylase is also found in T. maritima MSB8 * Corresponding author.
E-mail address: [email protected] (H.D. Yun). 0923-2508/$ – see front matter 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.09.005
cells only in extremely low amounts [9]. Since starch is rare in geothermal heated marine sediment, the presence of α-amylases in microorganisms living in these ecological niches is rather surprising. Although most amylases described to date are extracellular, recent work has described the detection of intracellular amylases [4,10,17]. However, little is known about intracellular amylolytic enzymes from either hyperthermophilic bacteria or archaea. Here we report the cloning and characterization of a gene encoding an intracellular α-amylase from T. maritima MSB8 in Escherichia coli. We also report biochemical characterization of the enzyme.
2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli XL1-Blue and recombinant E. coli harboring amyB gene were cultured in LB medium containing the appropriate antibiotics, ampicillin (50 µg/ml) or tetracycline
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(10 µg/ml). Genomic DNA of T. maritima MSB8 [12] was provided from Dr. Karl O. Stetter (Regensburg, Germany). 2.2. Recombinant DNA techniques Standard procedures for restriction endonuclease digestions, agarose gel electrophoresis, purification of DNA from agarose gels, DNA ligation, and other cloning related techniques were followed as described by Sambrook et al. [15]. Nucleotide sequences were determined with the dideoxychain termination method using the PRISM Ready Reaction Dye terminator/primer cycle sequencing kit (Perkin–Elmer Corp., Norwalk, CT, USA). 2.3. Cloning of the intracellular α-amylase gene T. maritima MSB8 was used as the DNA donor strain. The genomic library was constructed by ligating partially Sau3AI-digested genomic DNA fragments (3–5 kb) with pBluescript II SK+ cut with BamHI. To detect α-amylase activity from E. coli cells harboring the cloned α-amylase gene, bacterial colonies were first grown on LB broth supplemented with ampicillin (50 µg/ml). After incubation overnight, cells of 3 ml culture harvested by centrifugation at 5000 g for 5 min were suspended in 200 µl of 10 mM Tris– HCl buffer (pH 7.0) and sonicated. Supernatants obtained after centrifugation for 20 min at 17 000 g were used as crude enzymes. Crude enzymes were applied to LB agar supplemented with 0.1% (w/v) starch (Sigma Chemical Co., St. Louis, MO, USA). After incubation for 2 h, the plates were stained with Lugol’s solution (Merck KGaA, Darmstadt, Germany). Positive sonicated substances having intracellular α-amylase activities were surrounded by a white halo against the dark background. 2.4. Enzyme purification E. coli cells, harboring the cloned α-amylase gene, were cultivated in LB broth containing ampicillin (50 µg/ml) at 37 ◦ C for 16 h. The cells were harvested by centrifugation (6000 g, 10 min) and washed twice with 10 mM Tris– HCl buffer (pH 7.0). The cells were resuspended in the same buffer, disrupted by sonication at 4 ◦ C, and centrifuged (5000 g, 30 min) to remove cell debris. The supernatant was heat-treated at 70 ◦ C for 20 min, and the denatured host proteins were pelleted by centrifugation (17 000 g, 20 min). The α-amylase remained in the clear supernatant. Ammonium sulfate was added slowly to the resulting supernatant to 40% saturation. The precipitate that formed was removed by centrifugation at 17 000 g for 20 min. The enzyme solution was dialyzed and purified further by fast protein liquid chromatography using Q-Sepharose columns [6]. 2.5. Enzyme assay The α-amylase activity in the recombinant E. coli clones was determined by measuring the amount of reducing sugars
released during incubation with starch. A selected volume of enzyme was diluted with 10 mM Tris–HCl buffer (pH 7.0) to a total volume of 0.5 ml and was added to 250 µl of 1% (w/v) starch dissolved in 10 mM Tris–HCl buffer (pH 7.0), and the mixtures were incubated at different temperatures for 20 to 60 min. One unit of the enzyme activity was defined as the amount of the enzyme that liberated 1 µmol of reducing sugar per min at 70 ◦ C. The amounts of reducing sugars released were determined by the dinitrosalicylic acid method [1]. 2.6. Characterization of AmyB enzyme The effects of pH and temperature on the α-amylase activity were examined with the purified enzyme. The effect of the pH on the α-amylase activity was determined by using the protocol described above, to obtain values from pH 3.0 to 9.0; all of the assays were performed at 70 ◦ C. To determine the effect of temperature on the enzymatic activity, samples were incubated at temperatures from 30 to 90 ◦ C for 30 min. In all cases, the treatments were carried out in microcentrifuge tubes. After various time intervals, samples were withdrawn and clarified by centrifugation, and the enzyme activities were measured as described above. The effects of metal ions and other reagents in various concentrations on the α-amylase activity were measured as described above. Thermostability data were obtained by preincubating α-amylase samples in 10 mM Tris–HCl buffer (pH 7.0) at various temperatures and for various periods of time and measuring residual activity under described above assay condition. 2.7. Starch-nondenaturing PAGE and identification of gene product The nondenaturing polyacrylamide gel electrophoresis was carried out using the Bio-Rad Mini-PROTEIN 3 Cell electrophoresis unit (Bio-Rad, USA). Identification of an amyB gene product was performed in 0.75 mm gels in a vertical slab unit by a modification described by Park et al. [13]. The separating gel contained 10% acrylamide, 0.5% bisacrylamide, and 0.1% starch to detect the activity of electrophoresed α-amylase. The sonicated sample was mixed with sucrose–dye solution (50% sucrose, 0.1% bromophenol blue) in a ratio of 1:1 (v/v) and electrophoresed at 100 V and 4 ◦ C until the tracking dye migrated to the bottom of the gel. Active staining of the amylase activity in a nondenaturation PA gel was performed after washing the gel twice in 10 mM Tris–HCl buffer (pH 7.0). The gel was incubated in 10 mM Tris–HCl buffer (pH 7.0) at 50 ◦ C for 6 h. The acrylamide gel was stained with Lugol’s solution, which leaves a white halo zone where the starch has been degraded. We named this method “starch-nondenaturing PAGE”. Elution of the protein band containing the α-amylase was performed as described by Sá-Pereira et al. [16] after starch-nondenaturing
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PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method described by Laemmli [8]. The eluted protein was treated in sample buffer (62 mM Tris–HCl [pH 6.8], 10% glycerol, 0.025% bromophenol blue, 5% β-mercaptoethanol, and 2% SDS) at 95 ◦ C for 5 min before being loaded. The SDSPAGE standard, low range (Bio-Rad, USA) was used in order to determine the apparent molecular weight of the samples. Protein bands were visualized by staining with 0.2% Coomassie brilliant blue R-250.
3. Results
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alignment shown in Table 1. An identity matrix based on full alignments with α-amylases is presented in Table 2. It reveals that the T. maritima MSB8 α-amylase has about 12.5% identity to those of Bacillus halodurans (G84015), 12.0% to Natronococcus amylolyticus (BAA05516), 10.0% to Thermococcus hydrothermalis (AF068255), 10.0% to Pyrococcus furiosus (U96622), and 9.3% to Pyrococcus sp. KD01 (D83793). 3.4. Identification of the amyB product To facilitate the characterization of the α-amylase, we developed a direct activity staining technique (starch-nondenaturing PAGE) that allows rapid and specific detection of
3.1. amyB gene isolation and restriction map The amylase gene was isolated from a library of T. maritima MSB8 genomic DNA using a screen for amylase activity. One amylase activity clone carrying a 3.6-kb insert, pLY93, was further characterized (Figs. 1 and 2). Analysis of subclones of pLY93 revealed that the 2.1-kb EcoRI–ClaI fragment in pTMLY130 was sufficient to confer amylase activity in the plate assay. 3.2. Nucleotide sequence of the amyB gene Sequence analysis of the insert of pTMLY130 revealed one complete open reading frame of 1269 nucleotides that encodes a 422 amino acid protein with a predicted molecular mass of 50187 Da. We have named this gene amyB. Fig. 3 shows the amyB structural gene and the flanking regions (Accession No. AAD36717). 3.3. Comparison of AmyB of T. maritima MSB8 to the other known α-amylases Nakajima and his colleagues [11] identified four short primary sequence motifs, which are present in amylolytic enzymes that have other activities. These motifs are present in AmyB and are indicated as regions I to IV in the
Fig. 2. Detection of an α-amylase positive clone by starch agar diffusion method. The cell extracts were incubated at 37 ◦ C for 2 h. 1A, E. coli XL1-Blue as a negative control; 1B, E. coli XL1-Blue/pBluescript II SK+ as a negative control; 2A, E. coli XL1-Blue/pLY93; 2B, E. coli XL1-Blue/pTMLY100; 3A, E. coli XL1-Blue/pTMLY110; 3B, E. coli XL1-Blue/pTMLY120; 4A, E. coli XL1-Blue/pTMLY130; 4B, E. coli XL1-Blue/pTMLY140.
Fig. 1. Physical map of the T. maritima MSB8 intracellular α-amylase gene. The amyB ORF is shown by an open arrow. Cleavage sites by restriction enzymes BamHI, EcoRV, EcoRI, SacI, ClaI, and HindIII are shown. pLY93 was constructed by cloning a 3.6-kb Sau3AI fragment of T. maritima MSB8 into the BamHI site of the pBluescript II SK+ vector. pTMLY130 was derived by cloning the 2.1-kb EcoRI-ClaI fragment into the corresponding sites of that same plasmid vector.
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Fig. 3. Nucleotide and deduced amino acid sequence of the amyB gene. Initiation and termination codons are shaded. The ribosome binding site (RBS) is boxed, and four conserved regions are underlined.
Table 1 The four conserved regions found in the α-amylase family Source of enzyme T. maritima T. hydrothermalis P. furiosus Pyrococcus sp. KDO1 N. amylolyticus
Region I
Region II
Region III
Region IV
Assession No.a
DMVLNH D I V I NH DVV I NH D I V I NH D I VLNH * * **
G F RCDVAGL AWRFDYVKG GWRFDYVKG AWRFDYVKG G L R I DAAAH * *
EY V D EYWD EYWD EYWD EYWD ** *
FL ENHD FVANHD FVANHD FVANHD FVQNHD * ***
AAD36717 AF068255 U96622 D83793 BAA05516
Underlined sequences indicate amino acids which are identical to those found in T. maritime α-amylase. Asterisks indicate the invariant amino acids found on all α-amylases. a Accession No. from the SWISS-PROT protein and GenBank DNA sequence databases.
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Table 2 Percentage of sequence identity among α-amylases Accession No. AAD36717 G84015 BAA05516 D83793 U96622 AF068255
AAD36717
G84015
BAA05516
U96622
AF068255
D83793
100.0
12.5 100.0
12.0 11.4 100.0
10.0 10.7 25.3 100.0
10.0 10.1 25.3 82.1 100.0
9.3 10.5 25.3 85.7 82.7 100.0
T. maritima α-amylase (AAD36717), Bacillus halodurans (G84015), Thermococcus hydrothermalis α-amylase (AF068255), Pyrococcus furiosus α-amylase (U96622), Pyrococcus sp. KDO1 α-amylase (D83793), Natronococcus amylolyticus α-amylase (BAA05516).
corresponds well to the predicted AmyB molecular mass of 50 187 Da (Fig. 4B). 3.5. Purification of AmyB AmyB was purified from an E. coli AmyB overproducing strain as described in Section 2 using an assay that measures the release of reducing sugars. AmyB is thermostable. Hence, a key purification step was the heat treatment of the cell extract at 70 ◦ C for 20 min. AmyB did not lose any activity when incubated at 70 ◦ C for 20 min. After chromatography the α-amylase was purified 6-fold with a specific activity of 45 U/mg and a final yield of about 14%. Proteins from the purification steps were separated by SDS-PAGE, which indicated that only one protein band was present after this final purification step (data not shown). The size of the protein purified by this method was equivalent to that of the electroeluted protein identified by starch non-denaturing PAGE. 3.6. Characterization of AmyB
Fig. 4. (A) Detection of T. maritima α-amylase in E. coli by starch-nondenaturing-PAGE. E. coli XL1-Blue harboring pTMLY130 was grown in LB medium. The sonicated extract of the culture was loaded on a nondenaturing PA gel containing 0.1% soluble starch at 4 ◦ C. After electrophoresis and protein reaction, α-amylase activity was detected by staining with Lugol’s solution. (B) Estimation of the molecular weight of the α-amylase eluted from starch nondenaturing-PA gel by SDS-PAGE. The gel was stained with 0.025% Coomassie blue R-250. Molecular weight markers used were phosphorylase b (97 400), bovine serum albumin (66 200), ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500), and lysozyme (14 400).
α-amylase in a polyacrylamide slab gel. The technique takes advantage of the high specific activity of α-amylase against starch (we attempted to use starch-SDS-PAGE, but AmyB was not active under the conditions of that assay). This technique yielded a reproducible pattern of a white halo around a band where starch has been degraded (Fig. 4A). After starchnondenaturing-PAGE the band containing α-amylase activity was eluted by the method of electroelution described by Sá-Pereira et al. [16]. As determined by SDS-PAGE, this protein has a molecular mass of approximately 50 kD which
The effect of pH on the activity of the purified AmyB against starch was determined at 70 ◦ C in buffers ranging in pH from 3.0 to 9.0 (Fig. 5A). Maximal activity was observed at pH 7.0. Temperature dependence of AmyB activity toward starch was determined by measuring activity over a range of temperatures at pH 7.0. AmyB activity was dependent on temperature with maximal activity at 70 ◦ C (Fig. 5B). The amylase activity was measured at pH 7.0 and 70 ◦ C in the presence of some metal ions or various chemical reagents. Addition of EDTA (25 mM) and Triton X-100 (0.01%) resulted in inhibition. However, the addition of CaCl2 (10 mM) enhanced thermostability (Fig. 5C). Thermostability data were obtained by preincubating AmyB at various temperatures and then measuring residual starch hydrolyzing activity under the standard assay condition (pH 7.0). After 3 h of incubation at 80 ◦ C, the activity dropped by about 25% (Fig. 5D).
4. Discussion Hyperthermophiles are attracting growing attention because of the hyperthermophilicity of the organisms and their
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Fig. 5. (A) Effect of pH on the relative activity of AmyB. Enzyme activity was assayed at 70 ◦ C for 30 min at the indicated pH. (B) Effect of temperature on the relative activity of AmyB. Enzyme activity was assayed at pH 7.0 for 30 min at the indicated temperature. (C) Effect of metal ions and chemical reagents on the relative activity of AmyB. Enzyme activity was assayed at 70 ◦ C. (D) Effect of time and temperature on the relative activity of AmyB. Enzyme activity was assayed at 70 ◦ C, pH 7 for 180 min at the indicated reaction time.
enzymes. Amylolytic enzymes are among the best characterized and have been used to study hyperthermophilicity. Many extracellular amylolytic enzymes of T. maritima have been studied [2,7] but intracellular forms of the enzyme, like the AmyB protein from T. maritima MSB8, which we characterized and discuss in this paper, have not been reported previously. The predicted amino acid sequence of AmyB displays less than 10% identity to other α-amylases from other organisms (see Table 2). In T. maritima several other amylolytic enzymes are present in addition to AmyB, including TM1840 (AmyA/AAD36902) [9], TM1835 (cyclomaltodextrinase, putative/AAD36898), TM0364 (4-αglucanotransferase), TM0767 (maltodextrin glycosyltransferase/AAD35849), and TM1845 (pullulanase/AAD36907) [12]. Among the glycosyl hydrolases, amylases are grouped into two families, family 13 (mostly α-amylases) and family 57 [5]. Four T. maritima genes (TM1835, TM0364, TM0767, and TM1845) code for proteins that differ in length and domain topologies [http://www.ncbi.nlm.nih.gov (T. maritima complete genome/NC_000853)], which is not uncommon among family 13 members. The deduced amino acid sequence of AmyB had low identity with these other T. maritima amylolytic enzymes, sharing only about 5.8%
identity with TM1840, 16.9% with TM0364, 11.7% with TM0767, 15.1% with TM1835, and 5.8% with TM1845, respectively (data not shown). Interestingly, comparison of the primary sequence of AmyB described here with that of the extracellular α-amylase AmyA of T. maritima MSB8 described by Liebl et al. [9], revealed that these are two completely different enzymes. AmyB displays four highly conserved regions (see Table 1) present in α-amylases of other origins as for instance G84015 from Bacillus halodurans, which is a member of family 13. The catalytic residues of this family are located in those conserved regions 2, 3, and 4 [5]. Amylases are typically purified using two or more chromatographic steps. In the present work, we purified AmyB using a different methodology based on electrophoretic elution [16], which simplifies the purification of the amylase expressed in E. coli. After electrophoresis on polyacrylamide gels containing the amylase substrate, the gels were washed, incubated in buffer and immersed in staining solution. The protein conferring enzymatic activity was revealed as a white band and the amylase could be eluted from the gel slices by electroelution. The apparent molecular mass of the eluted protein was 50 kDa, which corresponds well with the predicted size of AmyB protein (50 187 Da). AmyB exibited
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optimum activity at pH 7.0 and 70 ◦ C and its thermostability was enhanced in the the presence of Ca2+ . The physiological role of intracellular amylases remains to be elucidated. They may be involved in breaking down glycogen-like intracellular polysaccharides, as has been suggested for the AmyA intracellular amylase of E. coli [14]. Many prokaryotes accumulate glycogen when their growth is nutrient-limited in the presence of an excess of carbon source. In some bacteria the genes for glycogen, biosynthesis and degradation are organized in an operon or cluster [20]. However, the genes that flank amyB in T. maritima MSB8 were not related to the glg operon. Nevertheless, the presence of an intracellular α-amylase indicates that this organism also has intracellular amylase substrates. Further work should allow unraveling the function and regulation of AmyB and related enzymes in T. maritima MSB8.
Acknowledgements We thank Dr. Karl O. Stetter (Regensburg, Germany) for providing the genomic DNA of T. maritima MSB8. This work was supported by grant 2000-1-22100-004-5 from the Basic Research Program of KOSEF and the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (Grant MG02-0101-0031-0-1) (H.D.Y.), Republic of Korea. W.J.L. is supported by scholarships from the BK21 Program, Ministry of Education and Human Resources Development, Korea.
References [1] P. Bernfeld, Amylases α and β, Methods Enzymol. 1 (1955) 149–155. [2] M. Bibel, C. Brettl, U. Gosslar, G. Kriegshäuser, W. Liebl, Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima, FEMS Microbiol. Lett. 158 (1998) 9–15. [3] J.M. Bragger, R.M. Daniel, T. Coolbear, H.W. Morgan, Very stable enzymes from extremely thermophilic archaebacteria and eubacteria, Appl. Microbiol. Biotechnol. 31 (1989) 556–561. [4] H.Y. Cho, Y.W. Kim, T.J. Kim, H.S. Lee, D.Y. Kim, J.W. Kim, Y.W. Lee, S.B. Lee, K.H. Park, Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2, Biochem. Biophys. Acta 1478 (2000) 333–340.
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[5] B. Henrissat, A. Bairoch, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316 (1996) 695–696. [6] R. Huber, K.O. Stetter, The Prokaryotes, Springer-Verlag, Berlin, 1992, pp. 3809–3815. [7] W. Huber, W. Liebl, Acceptor specificity of 4-α-glucanotransferase of Thermotoga maritima, Biocatalysis 11 (1994) 105–115. [8] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 , Nature 277 (1970) 680–685. [9] W. Liebl, I. Stemplinger, P. Ruile, Properties and gene structure of the Thermotoga maritima α-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium, J. Bacteriol. 179 (1997) 941–948. [10] W.J. Lim, S.R. Park, S.J. Cho, M.K. Kim, S.K. Ryu, S.Y. Hong, W.T. Seo, H. Kim, H.D. Yun, Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35, Biochem. Biophys. Res. Commun. 287 (2001) 348–354. [11] R. Nakajima, T. Imanaka, S. Aiba, Comparison of amino acid sequences of eleven different α-amylases, Appl. Microbiol. Biotechnol. 23 (1986) 355–360. [12] K.E. Nelson, R.A. Clayton, S.R. Gill, M.L. Gwinn, R.J. Dodson, D.H. Haft, E.K. Hickey, Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritime, Nature 399 (1999) 323–329. [13] S.R. Park, M.K. Kim, J.O. Kim, D.W. Bae, S.J. Cho, Y.U. Cho, H.D. Yun, Characterization of Erwinia chrysanthemi PY35 cel and pel gene existing in tandem and rapid identification of their gene products, Biochem. Biophys. Res. Commun. 268 (2000) 420–425. [14] M. Raha, I. Kawagishi, V. Müller, M. Kihara, R.M. Macnab, Escherichia coli produces a cytoplasmic α-amylase, AmyA, J. Bacteriol. 174 (1992) 6644–6652. [15] J. Sambrook, D.W. Russell, Molecular Cloning, A Laboratory Manual, 3th Edition, Cold Spring Harbor Laboratory Press, NY, 2001. [16] P. Sá-Pereira, J. Duarte, M. Costa-Ferreira, Electroelution as a simple and fast protein purification method, isolation of an extracellular xylanase from Bacillus sp. CCMI 966, Enzyme Microb. Technol. 27 (2000) 95–99. [17] E. Satoh, T. Uchimura, T. Kudo, K. Komagata, Purification, characterization, and nucleotide sequence of an intracellular maltotrioseproducing α-amylase from Streptococcus bovis 148, Appl. Environ. Microbiol. 63 (1997) 4941–4944. [18] J. Schumann, A. Wrba, R. Jaenicke, K.O. Stetter, Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritime, FEBS Lett. 282 (1991) 122–126. [19] K.O. Stetter, Life at the upper temperature border, in: J. Van, K.T.T. Van, J.C. Mounolou, J. Schneider, C. McKay (Eds.), Colloque Interdisciplinaire du Comite National de la Recherche Scientifique, Frontiers of Life, Editions Frontieres, Gif-sur-Yvette, 1992, pp. 195– 219. [20] J.E. Ugalde, V. Lepek, A. Uttaro, J. Estrella, A. Iglesias, R.A. Ugalde, Gene organization and transcription analysis of the Agrobacterium tumefaciens glycogen (glg) operon: Two transcripts for the single phosphoglucomutase gene, J. Bacteriol. 180 (1998) 6557–6564.
Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8 Woo Jin Lim a , Sang Ryeol Park a , Chang Long An a , Jong Yeoul Lee a , Su Young Hong a , Eun Chule Shin a , Eun Ju Kim a , Jong Ok Kim c , Hoon Kim d , Han Dae Yun a,b,∗ a Division of Applied Life Science, Gyeongsang National University, Chinju 660–701, Republic of Korea b Research Institute of Life Science, Gyeongsang National University, Chinju 660–701, Republic of Korea c Takara Korea Biomedical Inc. R & D Center, Uiwang 437–020, Republic of Korea d Department of Agricultural Chemistry, Sunchon National University, Sunchon 540–742, Republic of Korea
Received 10 February 2003; accepted 5 September 2003 First published online 9 September 2003
Abstract The gene encoding an intracellular α-amylase, AmyB (TM1650), from Thermotoga maritima MSB8, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli. The AmyB enzyme hydrolyzed α-1,4 starch linkage. The amyB gene is 1269 bp in length, encoding a protein of 422 amino acids (calculated molecular mass of 50 187 Da). The molecular weight of the enzyme was estimated to be 50 000 Da by SDS-PAGE after starch-nondenaturing-PAGE. The amino acid sequence of AmyB showed less than 12% identity to other amylases, but contained four regions that are highly conserved among α-amylases. The AmyB α-amylase exhibited maximal enzymatic activity at pH 7.0 and its optimum temperature for activity was 70 ◦ C. Like the α-amylases of many other organisms, the thermostability of T. maritima MSB8 α-amylase, AmyB expressed in E. coli was enhanced in the presence of Ca2+ (10 mM). 2003 Elsevier SAS. All rights reserved. Keywords: Thermotoga maritima; Intracellular α-amylase; Starch-nondenaturing PAGE
1. Introduction Hyperthermophilic bacteria and archaea, which grow optimally above 80 ◦ C, are of considerable interest because of the hyperthermostability of their enzymes [19]. In industry, hyperthermophilic enzymes could be advantageous not only for their extreme thermostability but also because of their resistance to denaturing agents, solvents, and proteolytic enzymes [3]. The archaea Thermatoga maritima is capable of utilizing simple carbohydrates as well as complex polysaccharides as energy sources. These include glucose, sucrose, cellulose, xylan and starch, the carbohydrate of interest here [6]. Schumann et al. [18] reported the existence of hydrolytic starch-degrading activities in T. maritima, but the activity levels seemed too low to allow purification and characterization. α-Amylase is also found in T. maritima MSB8 * Corresponding author.
E-mail address: [email protected] (H.D. Yun). 0923-2508/$ – see front matter 2003 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2003.09.005
cells only in extremely low amounts [9]. Since starch is rare in geothermal heated marine sediment, the presence of α-amylases in microorganisms living in these ecological niches is rather surprising. Although most amylases described to date are extracellular, recent work has described the detection of intracellular amylases [4,10,17]. However, little is known about intracellular amylolytic enzymes from either hyperthermophilic bacteria or archaea. Here we report the cloning and characterization of a gene encoding an intracellular α-amylase from T. maritima MSB8 in Escherichia coli. We also report biochemical characterization of the enzyme.
2. Materials and methods 2.1. Bacterial strains and growth conditions E. coli XL1-Blue and recombinant E. coli harboring amyB gene were cultured in LB medium containing the appropriate antibiotics, ampicillin (50 µg/ml) or tetracycline
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(10 µg/ml). Genomic DNA of T. maritima MSB8 [12] was provided from Dr. Karl O. Stetter (Regensburg, Germany). 2.2. Recombinant DNA techniques Standard procedures for restriction endonuclease digestions, agarose gel electrophoresis, purification of DNA from agarose gels, DNA ligation, and other cloning related techniques were followed as described by Sambrook et al. [15]. Nucleotide sequences were determined with the dideoxychain termination method using the PRISM Ready Reaction Dye terminator/primer cycle sequencing kit (Perkin–Elmer Corp., Norwalk, CT, USA). 2.3. Cloning of the intracellular α-amylase gene T. maritima MSB8 was used as the DNA donor strain. The genomic library was constructed by ligating partially Sau3AI-digested genomic DNA fragments (3–5 kb) with pBluescript II SK+ cut with BamHI. To detect α-amylase activity from E. coli cells harboring the cloned α-amylase gene, bacterial colonies were first grown on LB broth supplemented with ampicillin (50 µg/ml). After incubation overnight, cells of 3 ml culture harvested by centrifugation at 5000 g for 5 min were suspended in 200 µl of 10 mM Tris– HCl buffer (pH 7.0) and sonicated. Supernatants obtained after centrifugation for 20 min at 17 000 g were used as crude enzymes. Crude enzymes were applied to LB agar supplemented with 0.1% (w/v) starch (Sigma Chemical Co., St. Louis, MO, USA). After incubation for 2 h, the plates were stained with Lugol’s solution (Merck KGaA, Darmstadt, Germany). Positive sonicated substances having intracellular α-amylase activities were surrounded by a white halo against the dark background. 2.4. Enzyme purification E. coli cells, harboring the cloned α-amylase gene, were cultivated in LB broth containing ampicillin (50 µg/ml) at 37 ◦ C for 16 h. The cells were harvested by centrifugation (6000 g, 10 min) and washed twice with 10 mM Tris– HCl buffer (pH 7.0). The cells were resuspended in the same buffer, disrupted by sonication at 4 ◦ C, and centrifuged (5000 g, 30 min) to remove cell debris. The supernatant was heat-treated at 70 ◦ C for 20 min, and the denatured host proteins were pelleted by centrifugation (17 000 g, 20 min). The α-amylase remained in the clear supernatant. Ammonium sulfate was added slowly to the resulting supernatant to 40% saturation. The precipitate that formed was removed by centrifugation at 17 000 g for 20 min. The enzyme solution was dialyzed and purified further by fast protein liquid chromatography using Q-Sepharose columns [6]. 2.5. Enzyme assay The α-amylase activity in the recombinant E. coli clones was determined by measuring the amount of reducing sugars
released during incubation with starch. A selected volume of enzyme was diluted with 10 mM Tris–HCl buffer (pH 7.0) to a total volume of 0.5 ml and was added to 250 µl of 1% (w/v) starch dissolved in 10 mM Tris–HCl buffer (pH 7.0), and the mixtures were incubated at different temperatures for 20 to 60 min. One unit of the enzyme activity was defined as the amount of the enzyme that liberated 1 µmol of reducing sugar per min at 70 ◦ C. The amounts of reducing sugars released were determined by the dinitrosalicylic acid method [1]. 2.6. Characterization of AmyB enzyme The effects of pH and temperature on the α-amylase activity were examined with the purified enzyme. The effect of the pH on the α-amylase activity was determined by using the protocol described above, to obtain values from pH 3.0 to 9.0; all of the assays were performed at 70 ◦ C. To determine the effect of temperature on the enzymatic activity, samples were incubated at temperatures from 30 to 90 ◦ C for 30 min. In all cases, the treatments were carried out in microcentrifuge tubes. After various time intervals, samples were withdrawn and clarified by centrifugation, and the enzyme activities were measured as described above. The effects of metal ions and other reagents in various concentrations on the α-amylase activity were measured as described above. Thermostability data were obtained by preincubating α-amylase samples in 10 mM Tris–HCl buffer (pH 7.0) at various temperatures and for various periods of time and measuring residual activity under described above assay condition. 2.7. Starch-nondenaturing PAGE and identification of gene product The nondenaturing polyacrylamide gel electrophoresis was carried out using the Bio-Rad Mini-PROTEIN 3 Cell electrophoresis unit (Bio-Rad, USA). Identification of an amyB gene product was performed in 0.75 mm gels in a vertical slab unit by a modification described by Park et al. [13]. The separating gel contained 10% acrylamide, 0.5% bisacrylamide, and 0.1% starch to detect the activity of electrophoresed α-amylase. The sonicated sample was mixed with sucrose–dye solution (50% sucrose, 0.1% bromophenol blue) in a ratio of 1:1 (v/v) and electrophoresed at 100 V and 4 ◦ C until the tracking dye migrated to the bottom of the gel. Active staining of the amylase activity in a nondenaturation PA gel was performed after washing the gel twice in 10 mM Tris–HCl buffer (pH 7.0). The gel was incubated in 10 mM Tris–HCl buffer (pH 7.0) at 50 ◦ C for 6 h. The acrylamide gel was stained with Lugol’s solution, which leaves a white halo zone where the starch has been degraded. We named this method “starch-nondenaturing PAGE”. Elution of the protein band containing the α-amylase was performed as described by Sá-Pereira et al. [16] after starch-nondenaturing
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PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the method described by Laemmli [8]. The eluted protein was treated in sample buffer (62 mM Tris–HCl [pH 6.8], 10% glycerol, 0.025% bromophenol blue, 5% β-mercaptoethanol, and 2% SDS) at 95 ◦ C for 5 min before being loaded. The SDSPAGE standard, low range (Bio-Rad, USA) was used in order to determine the apparent molecular weight of the samples. Protein bands were visualized by staining with 0.2% Coomassie brilliant blue R-250.
3. Results
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alignment shown in Table 1. An identity matrix based on full alignments with α-amylases is presented in Table 2. It reveals that the T. maritima MSB8 α-amylase has about 12.5% identity to those of Bacillus halodurans (G84015), 12.0% to Natronococcus amylolyticus (BAA05516), 10.0% to Thermococcus hydrothermalis (AF068255), 10.0% to Pyrococcus furiosus (U96622), and 9.3% to Pyrococcus sp. KD01 (D83793). 3.4. Identification of the amyB product To facilitate the characterization of the α-amylase, we developed a direct activity staining technique (starch-nondenaturing PAGE) that allows rapid and specific detection of
3.1. amyB gene isolation and restriction map The amylase gene was isolated from a library of T. maritima MSB8 genomic DNA using a screen for amylase activity. One amylase activity clone carrying a 3.6-kb insert, pLY93, was further characterized (Figs. 1 and 2). Analysis of subclones of pLY93 revealed that the 2.1-kb EcoRI–ClaI fragment in pTMLY130 was sufficient to confer amylase activity in the plate assay. 3.2. Nucleotide sequence of the amyB gene Sequence analysis of the insert of pTMLY130 revealed one complete open reading frame of 1269 nucleotides that encodes a 422 amino acid protein with a predicted molecular mass of 50187 Da. We have named this gene amyB. Fig. 3 shows the amyB structural gene and the flanking regions (Accession No. AAD36717). 3.3. Comparison of AmyB of T. maritima MSB8 to the other known α-amylases Nakajima and his colleagues [11] identified four short primary sequence motifs, which are present in amylolytic enzymes that have other activities. These motifs are present in AmyB and are indicated as regions I to IV in the
Fig. 2. Detection of an α-amylase positive clone by starch agar diffusion method. The cell extracts were incubated at 37 ◦ C for 2 h. 1A, E. coli XL1-Blue as a negative control; 1B, E. coli XL1-Blue/pBluescript II SK+ as a negative control; 2A, E. coli XL1-Blue/pLY93; 2B, E. coli XL1-Blue/pTMLY100; 3A, E. coli XL1-Blue/pTMLY110; 3B, E. coli XL1-Blue/pTMLY120; 4A, E. coli XL1-Blue/pTMLY130; 4B, E. coli XL1-Blue/pTMLY140.
Fig. 1. Physical map of the T. maritima MSB8 intracellular α-amylase gene. The amyB ORF is shown by an open arrow. Cleavage sites by restriction enzymes BamHI, EcoRV, EcoRI, SacI, ClaI, and HindIII are shown. pLY93 was constructed by cloning a 3.6-kb Sau3AI fragment of T. maritima MSB8 into the BamHI site of the pBluescript II SK+ vector. pTMLY130 was derived by cloning the 2.1-kb EcoRI-ClaI fragment into the corresponding sites of that same plasmid vector.
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Fig. 3. Nucleotide and deduced amino acid sequence of the amyB gene. Initiation and termination codons are shaded. The ribosome binding site (RBS) is boxed, and four conserved regions are underlined.
Table 1 The four conserved regions found in the α-amylase family Source of enzyme T. maritima T. hydrothermalis P. furiosus Pyrococcus sp. KDO1 N. amylolyticus
Region I
Region II
Region III
Region IV
Assession No.a
DMVLNH D I V I NH DVV I NH D I V I NH D I VLNH * * **
G F RCDVAGL AWRFDYVKG GWRFDYVKG AWRFDYVKG G L R I DAAAH * *
EY V D EYWD EYWD EYWD EYWD ** *
FL ENHD FVANHD FVANHD FVANHD FVQNHD * ***
AAD36717 AF068255 U96622 D83793 BAA05516
Underlined sequences indicate amino acids which are identical to those found in T. maritime α-amylase. Asterisks indicate the invariant amino acids found on all α-amylases. a Accession No. from the SWISS-PROT protein and GenBank DNA sequence databases.
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Table 2 Percentage of sequence identity among α-amylases Accession No. AAD36717 G84015 BAA05516 D83793 U96622 AF068255
AAD36717
G84015
BAA05516
U96622
AF068255
D83793
100.0
12.5 100.0
12.0 11.4 100.0
10.0 10.7 25.3 100.0
10.0 10.1 25.3 82.1 100.0
9.3 10.5 25.3 85.7 82.7 100.0
T. maritima α-amylase (AAD36717), Bacillus halodurans (G84015), Thermococcus hydrothermalis α-amylase (AF068255), Pyrococcus furiosus α-amylase (U96622), Pyrococcus sp. KDO1 α-amylase (D83793), Natronococcus amylolyticus α-amylase (BAA05516).
corresponds well to the predicted AmyB molecular mass of 50 187 Da (Fig. 4B). 3.5. Purification of AmyB AmyB was purified from an E. coli AmyB overproducing strain as described in Section 2 using an assay that measures the release of reducing sugars. AmyB is thermostable. Hence, a key purification step was the heat treatment of the cell extract at 70 ◦ C for 20 min. AmyB did not lose any activity when incubated at 70 ◦ C for 20 min. After chromatography the α-amylase was purified 6-fold with a specific activity of 45 U/mg and a final yield of about 14%. Proteins from the purification steps were separated by SDS-PAGE, which indicated that only one protein band was present after this final purification step (data not shown). The size of the protein purified by this method was equivalent to that of the electroeluted protein identified by starch non-denaturing PAGE. 3.6. Characterization of AmyB
Fig. 4. (A) Detection of T. maritima α-amylase in E. coli by starch-nondenaturing-PAGE. E. coli XL1-Blue harboring pTMLY130 was grown in LB medium. The sonicated extract of the culture was loaded on a nondenaturing PA gel containing 0.1% soluble starch at 4 ◦ C. After electrophoresis and protein reaction, α-amylase activity was detected by staining with Lugol’s solution. (B) Estimation of the molecular weight of the α-amylase eluted from starch nondenaturing-PA gel by SDS-PAGE. The gel was stained with 0.025% Coomassie blue R-250. Molecular weight markers used were phosphorylase b (97 400), bovine serum albumin (66 200), ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500), and lysozyme (14 400).
α-amylase in a polyacrylamide slab gel. The technique takes advantage of the high specific activity of α-amylase against starch (we attempted to use starch-SDS-PAGE, but AmyB was not active under the conditions of that assay). This technique yielded a reproducible pattern of a white halo around a band where starch has been degraded (Fig. 4A). After starchnondenaturing-PAGE the band containing α-amylase activity was eluted by the method of electroelution described by Sá-Pereira et al. [16]. As determined by SDS-PAGE, this protein has a molecular mass of approximately 50 kD which
The effect of pH on the activity of the purified AmyB against starch was determined at 70 ◦ C in buffers ranging in pH from 3.0 to 9.0 (Fig. 5A). Maximal activity was observed at pH 7.0. Temperature dependence of AmyB activity toward starch was determined by measuring activity over a range of temperatures at pH 7.0. AmyB activity was dependent on temperature with maximal activity at 70 ◦ C (Fig. 5B). The amylase activity was measured at pH 7.0 and 70 ◦ C in the presence of some metal ions or various chemical reagents. Addition of EDTA (25 mM) and Triton X-100 (0.01%) resulted in inhibition. However, the addition of CaCl2 (10 mM) enhanced thermostability (Fig. 5C). Thermostability data were obtained by preincubating AmyB at various temperatures and then measuring residual starch hydrolyzing activity under the standard assay condition (pH 7.0). After 3 h of incubation at 80 ◦ C, the activity dropped by about 25% (Fig. 5D).
4. Discussion Hyperthermophiles are attracting growing attention because of the hyperthermophilicity of the organisms and their
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Fig. 5. (A) Effect of pH on the relative activity of AmyB. Enzyme activity was assayed at 70 ◦ C for 30 min at the indicated pH. (B) Effect of temperature on the relative activity of AmyB. Enzyme activity was assayed at pH 7.0 for 30 min at the indicated temperature. (C) Effect of metal ions and chemical reagents on the relative activity of AmyB. Enzyme activity was assayed at 70 ◦ C. (D) Effect of time and temperature on the relative activity of AmyB. Enzyme activity was assayed at 70 ◦ C, pH 7 for 180 min at the indicated reaction time.
enzymes. Amylolytic enzymes are among the best characterized and have been used to study hyperthermophilicity. Many extracellular amylolytic enzymes of T. maritima have been studied [2,7] but intracellular forms of the enzyme, like the AmyB protein from T. maritima MSB8, which we characterized and discuss in this paper, have not been reported previously. The predicted amino acid sequence of AmyB displays less than 10% identity to other α-amylases from other organisms (see Table 2). In T. maritima several other amylolytic enzymes are present in addition to AmyB, including TM1840 (AmyA/AAD36902) [9], TM1835 (cyclomaltodextrinase, putative/AAD36898), TM0364 (4-αglucanotransferase), TM0767 (maltodextrin glycosyltransferase/AAD35849), and TM1845 (pullulanase/AAD36907) [12]. Among the glycosyl hydrolases, amylases are grouped into two families, family 13 (mostly α-amylases) and family 57 [5]. Four T. maritima genes (TM1835, TM0364, TM0767, and TM1845) code for proteins that differ in length and domain topologies [http://www.ncbi.nlm.nih.gov (T. maritima complete genome/NC_000853)], which is not uncommon among family 13 members. The deduced amino acid sequence of AmyB had low identity with these other T. maritima amylolytic enzymes, sharing only about 5.8%
identity with TM1840, 16.9% with TM0364, 11.7% with TM0767, 15.1% with TM1835, and 5.8% with TM1845, respectively (data not shown). Interestingly, comparison of the primary sequence of AmyB described here with that of the extracellular α-amylase AmyA of T. maritima MSB8 described by Liebl et al. [9], revealed that these are two completely different enzymes. AmyB displays four highly conserved regions (see Table 1) present in α-amylases of other origins as for instance G84015 from Bacillus halodurans, which is a member of family 13. The catalytic residues of this family are located in those conserved regions 2, 3, and 4 [5]. Amylases are typically purified using two or more chromatographic steps. In the present work, we purified AmyB using a different methodology based on electrophoretic elution [16], which simplifies the purification of the amylase expressed in E. coli. After electrophoresis on polyacrylamide gels containing the amylase substrate, the gels were washed, incubated in buffer and immersed in staining solution. The protein conferring enzymatic activity was revealed as a white band and the amylase could be eluted from the gel slices by electroelution. The apparent molecular mass of the eluted protein was 50 kDa, which corresponds well with the predicted size of AmyB protein (50 187 Da). AmyB exibited
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optimum activity at pH 7.0 and 70 ◦ C and its thermostability was enhanced in the the presence of Ca2+ . The physiological role of intracellular amylases remains to be elucidated. They may be involved in breaking down glycogen-like intracellular polysaccharides, as has been suggested for the AmyA intracellular amylase of E. coli [14]. Many prokaryotes accumulate glycogen when their growth is nutrient-limited in the presence of an excess of carbon source. In some bacteria the genes for glycogen, biosynthesis and degradation are organized in an operon or cluster [20]. However, the genes that flank amyB in T. maritima MSB8 were not related to the glg operon. Nevertheless, the presence of an intracellular α-amylase indicates that this organism also has intracellular amylase substrates. Further work should allow unraveling the function and regulation of AmyB and related enzymes in T. maritima MSB8.
Acknowledgements We thank Dr. Karl O. Stetter (Regensburg, Germany) for providing the genomic DNA of T. maritima MSB8. This work was supported by grant 2000-1-22100-004-5 from the Basic Research Program of KOSEF and the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (Grant MG02-0101-0031-0-1) (H.D.Y.), Republic of Korea. W.J.L. is supported by scholarships from the BK21 Program, Ministry of Education and Human Resources Development, Korea.
References [1] P. Bernfeld, Amylases α and β, Methods Enzymol. 1 (1955) 149–155. [2] M. Bibel, C. Brettl, U. Gosslar, G. Kriegshäuser, W. Liebl, Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima, FEMS Microbiol. Lett. 158 (1998) 9–15. [3] J.M. Bragger, R.M. Daniel, T. Coolbear, H.W. Morgan, Very stable enzymes from extremely thermophilic archaebacteria and eubacteria, Appl. Microbiol. Biotechnol. 31 (1989) 556–561. [4] H.Y. Cho, Y.W. Kim, T.J. Kim, H.S. Lee, D.Y. Kim, J.W. Kim, Y.W. Lee, S.B. Lee, K.H. Park, Molecular characterization of a dimeric intracellular maltogenic amylase of Bacillus subtilis SUH4-2, Biochem. Biophys. Acta 1478 (2000) 333–340.
687
[5] B. Henrissat, A. Bairoch, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316 (1996) 695–696. [6] R. Huber, K.O. Stetter, The Prokaryotes, Springer-Verlag, Berlin, 1992, pp. 3809–3815. [7] W. Huber, W. Liebl, Acceptor specificity of 4-α-glucanotransferase of Thermotoga maritima, Biocatalysis 11 (1994) 105–115. [8] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4 , Nature 277 (1970) 680–685. [9] W. Liebl, I. Stemplinger, P. Ruile, Properties and gene structure of the Thermotoga maritima α-amylase AmyA, a putative lipoprotein of a hyperthermophilic bacterium, J. Bacteriol. 179 (1997) 941–948. [10] W.J. Lim, S.R. Park, S.J. Cho, M.K. Kim, S.K. Ryu, S.Y. Hong, W.T. Seo, H. Kim, H.D. Yun, Cloning and characterization of an intracellular isoamylase gene from Pectobacterium chrysanthemi PY35, Biochem. Biophys. Res. Commun. 287 (2001) 348–354. [11] R. Nakajima, T. Imanaka, S. Aiba, Comparison of amino acid sequences of eleven different α-amylases, Appl. Microbiol. Biotechnol. 23 (1986) 355–360. [12] K.E. Nelson, R.A. Clayton, S.R. Gill, M.L. Gwinn, R.J. Dodson, D.H. Haft, E.K. Hickey, Evidence for lateral gene transfer between archaea and bacteria from genome sequence of Thermotoga maritime, Nature 399 (1999) 323–329. [13] S.R. Park, M.K. Kim, J.O. Kim, D.W. Bae, S.J. Cho, Y.U. Cho, H.D. Yun, Characterization of Erwinia chrysanthemi PY35 cel and pel gene existing in tandem and rapid identification of their gene products, Biochem. Biophys. Res. Commun. 268 (2000) 420–425. [14] M. Raha, I. Kawagishi, V. Müller, M. Kihara, R.M. Macnab, Escherichia coli produces a cytoplasmic α-amylase, AmyA, J. Bacteriol. 174 (1992) 6644–6652. [15] J. Sambrook, D.W. Russell, Molecular Cloning, A Laboratory Manual, 3th Edition, Cold Spring Harbor Laboratory Press, NY, 2001. [16] P. Sá-Pereira, J. Duarte, M. Costa-Ferreira, Electroelution as a simple and fast protein purification method, isolation of an extracellular xylanase from Bacillus sp. CCMI 966, Enzyme Microb. Technol. 27 (2000) 95–99. [17] E. Satoh, T. Uchimura, T. Kudo, K. Komagata, Purification, characterization, and nucleotide sequence of an intracellular maltotrioseproducing α-amylase from Streptococcus bovis 148, Appl. Environ. Microbiol. 63 (1997) 4941–4944. [18] J. Schumann, A. Wrba, R. Jaenicke, K.O. Stetter, Topographical and enzymatic characterization of amylases from the extremely thermophilic eubacterium Thermotoga maritime, FEBS Lett. 282 (1991) 122–126. [19] K.O. Stetter, Life at the upper temperature border, in: J. Van, K.T.T. Van, J.C. Mounolou, J. Schneider, C. McKay (Eds.), Colloque Interdisciplinaire du Comite National de la Recherche Scientifique, Frontiers of Life, Editions Frontieres, Gif-sur-Yvette, 1992, pp. 195– 219. [20] J.E. Ugalde, V. Lepek, A. Uttaro, J. Estrella, A. Iglesias, R.A. Ugalde, Gene organization and transcription analysis of the Agrobacterium tumefaciens glycogen (glg) operon: Two transcripts for the single phosphoglucomutase gene, J. Bacteriol. 180 (1998) 6557–6564.