Microbial Discovery Research Unit, School of Biomolecular and Biomedical Sciences, Faculty of Science, Griffith University, Brisbane, Queensland 4111, Australia1
Author for correspondence: Bharat Patel. Tel: +61 438 173 185. Fax: +61 7 3875 7800. e-mail: bharat{at}genomes.sci.gu.edu.au
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ABSTRACT |
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Keywords: alpha-amylase, halophile, thermophile, anaerobe, Halothermothrix orenii
Abbreviations: DNS, dinitrosalicylic acid
a The GenBank accession number for the sequence reported in this paper is AF442961.
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INTRODUCTION |
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Halothermothrix orenii (Cayol et al., 1994 ) and Thermohalobacter barriensis (Cayol et al., 2000
) are the only truly thermophilic halophilic prokaryotes that have been reported to date. Here, we report on an
-amylase cloned from H. orenii. H. orenii is an anaerobic bacterium that is thermophilic (optimum growth at 60 °C) and also a moderate halophile (optimum growth at 10%, w/v, NaCl) (Cayol et al., 1994
). It may therefore be a potential source for enzymes that are uniquely adapted to activity at high temperatures and salt concentrations. We have isolated a gene encoding an
-amylase from a Halothermothrix orenii genomic DNA library constructed in Escherichia coli. The enzyme was expressed with an N-terminal hexahistidine tag, purified and biochemically characterized. To our knowledge this is the first report of cloning or characterization of an extracellular enzyme from an anaerobic, moderately halophilic, thermophilic bacterium.
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METHODS |
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Screening of H. orenii genomic DNA library for starch-degrading activity.
Culturing of H. orenii and genomic DNA library construction were as described previously (Mijts & Patel, 2001 ). The H. orenii genomic library, which had a mean insert size of 3·5 kb and had been stored in 96-well microtitre plates, was replica-plated on to LB agar plates containing 0·3% (w/v) soluble starch and 100 µg ampicillin ml-1 using a 48-colony arrayer, grown at 37 °C for 18 h, sealed in plastic bags and transferred to a 65 °C incubator overnight. Starch-hydrolysing clones were detected by flooding plates with 0·2% (w/v) KI/0·1% (w/v) I2 solution and checking for halo formation.
Sequencing and identification of the amyA gene of H. orenii.
The complete double-stranded nucleotide sequence of the starch-degrading recombinant insert was determined using a primer-walking strategy with an ABI 377 automated DNA sequencer (ABI-Perkin Elmer) at the Griffith University Molecular Biology Facility. DNA sequencing reactions were performed as described previously (Mijts & Patel, 2001 ). Putative genes were identified using GeneMark software (Besemer & Borodovsky, 1999
) and function assigned by BLAST searches (Altschul et al., 1990
) against the GenBank nucleotide database (Benson et al., 2000
).
Construction of an amyA expression vector.
PCR amplification of the amyA coding sequence from the putative signal peptide cleavage site to a region downstream of the stop codon was carried out using the oligonucleotides F2-PstI (5'-TATCTGTTTTTCCTGTTTCTGCAGACGATTTCGAAAAAC) and R1-KpnI (5'-GCCTCGTGGAGGGTACCTATGACCTTG) with bases from the template sequence modified in order to generate restriction sites indicated as underlined bases. This PCR product was digested with KpnI followed by PstI, gel-purified, ligated with similarly treated pTrcHisB expression vector (Invitrogen) and transformed into electrocompetent cells of E. coli strain TOP10 (Invitrogen). A number of clones were sequenced using the primer-walking strategy described above and a clone with the correct sequence was used for all further expression and characterization experiments.
Expression and purification of recombinant AmyA.
Cultures (50 ml) of E. coli TOP10 cells containing a pTrcHis expression construct were grown overnight at 37 °C in LB Amp medium with and without 1 mM IPTG and harvested by centrifugation (5000 g for 10 min). Cells were resuspended in 5 ml native binding buffer (20 mM phosphate, 500 mM NaCl, pH 7·8), lysozyme was added to a final concentration of 100 µg ml-1 and the sample was incubated on ice for 15 min. The cell suspension was then sonicated at maximum intensity for three 10 s bursts while kept on ice. Final cell lysis was achieved by three rapid freeze/thaw cycles in liquid nitrogen and a 50 °C water bath. Any insoluble material was removed by centrifugation at 4000 g for 15 min. Heat precipitation (68 °C for 30 min) of the majority of host proteins in the presence of 0·5 M NaCl was used as a technique to partially purify AmyA expressed in E. coli. The protein preparation was then transferred to ice for 15 min to maximize E. coli protein precipitation and host proteins pelleted by centrifugation at 10000 g for 10 min; the supernatant was retained and stored at -20 °C. The partially purified, polyhisitidine-tagged recombinant enzyme was purified using ProBond columns (Invitrogen) according to the manufacturers instructions. Enzyme elution was performed using the imidazole elution protocol under native conditions.
Characterization of the recombinant amylase.
Unless otherwise indicated, the following standard assay was used. Predetermined units of AmyA were added to 50 µl Tris buffer pH 8·0, 5% (w/v) NaCl, 10 mM CaCl2 and 0·5% (w/v) starch in microtitre plates, which were incubated at 65 °C for 30 min in a Bio-Rad iCycler thermal cycler; the amount of starch hydrolysed was determined by using the starchiodine assay and/or the sugar released by using the DNS reducing sugar assay (described below). The substrate spectrum that could be used by the enzyme was determined by replacing starch with amylose, amylopectin, pullulan, glycogen and -, ß- and
-cyclodextrin. The pH optimum for enzyme was determined using the standard assay but with the following pH buffers (100 mM): Bistris/HCl (pH 5·57·0), Tris/HCl (pH 7·58·5) and glycine/NaOH (pH 8·510·5). The NaCl optimum for activity was determined using the standard assay described above but with NaCl at final concentrations up to 25% (w/v). Temperature optimum was determined using the standard assay in which the buffer was substituted with 100 mM Bistris/HCl pH 7·0 and the incubation temperatures ranged from 37 to 80 °C. The effect of NaCl and CaCl2 on enzyme thermostability was determined by preincubating enzyme solutions in 200 mM Tris buffer pH 8·0 at 70 °C and removing samples at 0, 60 and 120 min. Pre-incubation samples were set up at 0% (w/v) NaCl, 0 mM CaCl2; 10% (w/v) NaCl, 0 mM CaCl2; 0% (w/v) NaCl, 10 mM CaCl2; and 10% (w/v) NaCl, 10 mM CaCl2. Buffered starch/saline solution was then added to each sample to re-establish standard assay conditions and activity determined by reducing sugar assays after incubation at 65 °C for 30 min.
Enzyme digest time-course.
A starch digest time-course was performed by adding a suitable amount of enzyme to a 1500 µl solution [100 mM Tris pH 8·0, 5% (w/v) NaCl, 8 mM CaCl2, 0·5% (w/v) soluble starch], incubating at 65 °C and removing 150 µl aliquots for up 480 min for DNS reducing sugar assays (described below) and starchiodine assays. Reducing sugar production was calculated as a percentage of total starch conversion to sugars assuming maltose as an end product.
Transferase activity assay.
Transferase activity assays were adapted from a method developed to detect transferase activity in B. megaterium -amylase (Brumm et al., 1996
). Starch hydrolysis activity was measured using starchiodine assays under standard reaction conditions both with and without the addition of various mono-, di- and trisaccharides (fructose, galactose, glucose, maltose, lactose, sucrose, cellobiose, isomaltose, maltitol and maltotriose) at 0·1 M concentration. These carbohydrates are possible acceptor molecules in the transferase reaction and any transferase activity would increase the observed rate of starch hydrolysis.
Reducing sugar assay.
DNS assay reagent [50 µl; 1% (w/v) 1,3-dinitrosalicylic acid, 0·05% (w/v) sodium sulfite, 1% (w/v) sodium hydroxide, 10% (w/v) sodium potassium tartrate] was added to enzyme digests and the resulting samples were incubated at 98 °C for 10 min. Reducing sugar levels were then measured as A490 using a Wallac Victor 1420 Multilabel Counter. A series of maltose concentration standards was included with each reducing sugar assay. For experiments that required simultaneous incubation at various temperatures, reactions were performed in 0·2 ml microfuge tubes incubated in water baths at suitable temperatures. Reducing sugar levels were then determined as above.
Starchiodine assays.
Remnant starch was quantitatively determined by measuring the A650 of starchiodine complexes in solution. An iodine stock solution consisting of 0·3% (w/v) I2, 0·6% (w/v) KI was diluted 1/1000 in 17 mM acetic acid and 950 µl of this solution added to 50 µl of sample to be tested. Samples were mixed by vortexing and the A650 measured.
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RESULTS |
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Nucleotide sequence analysis of the amyA gene
Analysis of the insert DNA sequence with WebGeneMark heuristic approach software (Besemer & Borodovsky, 1999 ) indicated that the most probable translated open reading frame was from nucleotides 432 to 1977, beginning with a GTG start codon and preceded with a spacing of 8 nucleotides by a putative ribosome-binding site (5'-AAGGATG-3'). BLAST analysis indicated that this gene was homologous to bacterial
-amylase genes and it was designated amyA. Although other in-frame ATG and GTG start codons are present in this region these codons lack conserved ribosome-binding sites. A region that is extremely close to the bacterial promoter consensus sequence can be found upstream of the amyA gene. This sequence (TTGAAA-N17-TATAAT) begins at nucleotide 376 and differs from the consensus sequence by only one nucleotide.
Amino acid sequence analysis and comparison
The first 70 amino acid residues of the predicted ORF were analysed using SignalP version 2.0 signal peptide prediction software (Nielsen et al., 1997 ) trained on Gram-negative bacterial secretion signal peptide sequences. The results of this analysis indicated that a well-conserved Gram-negative signal peptide 25 amino acids in length was present. Assuming the signal peptide sequence is cleaved as predicted (VYANDF) the resulting polypeptide would be 490 amino acids in length with a molecular mass of 56965 kDa.
The derived amino acid sequence of amyA was used to perform a homology search using BLASTP software against the GenBank non-redundant database (Altschul et al., 1990 ). The results of this search indicated that AmyA showed high homology to a group of five enzymes:
-amylase from Bacillus megaterium (Metz et al., 1988
), neopullulanase from Paenibacillus polymyxa (Yebra et al., 1999
), periplasmic
-amylase from Xanthomonas campestris K-11151 (Abe et al., 1996
) and
-amylase AmyC from Dictyoglomus thermophilum (Horinouchi et al., 1988
). Lower levels of homology were also observed for other
-amylases, glucanotransferases and trehalose synthases from thermophilic bacteria.
The amino acid alignment of H. orenii AmyA with the five high-matching amino acid sequences of this group of enzymes, and showing the consensus regions commonly found in -amylases (Nakajima et al., 1986
) and also identified in the AmyA amino acid sequence, is presented in Fig. 1
. The members of this group share a number of unusual catalytic properties: (i) they are capable of hydrolysing cyclodextrins to varying degrees, despite lacking sequence homology to the pullulanase family, (ii) various degrees of transglycosylating activity are present in each of these enzymes, and (iii) some of the members catalyse the hydrolysis of pullulan to panose. Despite these curious properties, most of these enzymes have only been partially characterized, and in some cases the characterization was from crude protein extracts rather than from purifed proteins.
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The recombinant AmyA protein was purified from E. coli TOP10(pTH5A6) as described in Methods. The purification procedure is summarized in Table 1. The recombinant enzyme was judged homogeneous on the basis of SDS-PAGE (Fig. 2
). Very high levels of recombinant AmyA expression were observed in IPTG-induced cultures of TOP10(pTH5A6). Fig. 2
clearly indicates the high levels of AmyA expression observed in IPTG-induced cultures compared with non-induced controls. It is also clear that the majority of E. coli host proteins were removed in the heat-precipitation step. However, the additional step of hexahistidine tag affinity chromatography, though not necessary, was performed to ensure that absolute purity of the recombinant enzyme preparation was achieved.
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The purified AmyA was found to be stabilized at 70 °C by both NaCl and CaCl2 (Table 2). In the absence of both NaCl and CaCl2, activity was rapidly lost by incubation at 70 °C. Individually, 10% NaCl and 10 mM CaCl2 each improved the thermostability of the enzyme and optimal thermostability was observed when both 10% NaCl and 10 mM CaCl2 were present. AmyA was also found to be more stable in the presence of starch substrate (results not shown).
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DISCUSSION |
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Some enzymes with high amino acid homology to AmyA are able to hydrolyse unusual substrates such as pullulan and cyclodextrins to varying degrees. These activities are not present in AmyA, with activity being observed only on starch, amylose, amylopectin and glycogen. Also lacking in H. orenii AmyA was any transferase activity as detected in the closely related B. megaterium -amylase (Brumm et al., 1996
). No increase in AmyA starch hydrolysis reaction rates was observed in the presence of a variety of potential transferase acceptor saccharides. However, as for B. megaterium amylase, strong inhibition of starch-degrading activity by maltose and maltotriose was observed for H. orenii AmyA. The lack of transferase activity and activity on unusual substrates is less surprising considering the already considerable catalytic diversity observed within this relatively small cluster of enzymes. It is clear that relatively minor changes in important residues of these enzymes result in significant alterations in catalytic diversity.
Optimal activity for H. orenii AmyA was found to occur in conditions similar to those at which the source organism grows optimally in culture. The NaCl optimum for growth of H. orenii is around 10% (Cayol et al., 1994 ) and AmyA retained high levels of activity under these conditions. Although not as active or stable in the absence of NaCl, AmyA was still 44% active when NaCl was not present. Additionally, over 90% activity was observed at the remarkably high salt concentration of 25%. This type of extreme halotolerance has been observed in extracellular amylases from other halophilic organisms including the moderate halophiles Micrococcus halobius (Onishi & Sonoda, 1979
) and Halomonas meridiana (Coronado et al., 2000a
) and the extreme halophile Halobacterium halobium (Good & Hartman, 1970
). However, most enzymes from extreme halophilic Archaea, such as amylase from Natronococcus sp. strain Ah36, are completely unstable and inactive at submolar salt concentrations. AmyA was found to have an optimum temperature for starch hydrolysis of 65 °C. This is very close to the optimum growth temperature of Halothermothrix orenii of around 60 °C. AmyA was found to be relatively thermostable in the absence of starch when both CaCl2 and NaCl are present. CaCl2 binding has been reported to increase the overall structural integrity and thermal stability of
-amylases (Violet & Meunier, 1989
). AmyA had a pH optimum for activity of around pH 7·5. This is consistent with the optimum pH for growth of the source organism of around pH 7.
As with a previous report on randomly selected sequence tags generated from the H. orenii genomic library (Mijts & Patel, 2001 ), H. orenii AmyA also shows no significant excess of acidic amino acids. A significant acidic amino acid excess is a biochemical trait thought to be necessary for activity and stability of enzymes from some halophilic organisms. It is found in most enzymes from extreme halophiles of domain Archaea and in extracellular enzymes from moderate halophiles of domain Bacteria (Coronado et al., 2000b
). The extreme halotolerance of H. orenii AmyA suggests that this characteristic is not strictly necessary to maintain enzyme activity and stability at high salt concentrations.
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Received 23 November 2001;
revised 21 February 2002;
accepted 27 March 2002.
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