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Volume 272, Number 26,
Issue of June 27, 1997
pp. 16335-16342
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning, Sequencing, Characterization, and Expression of an
Extracellular -Amylase from the Hyperthermophilic Archaeon
Pyrococcus furiosus in Escherichia coli and
Bacillus subtilis*
(Received for publication, January 24, 1997)
Steen
Jørgensen
,
Constantin E.
Vorgias
§ and
Garabed
Antranikian
¶
From Novo Nordisk A/S, Enzyme Research, Bacterial
Gene Technology, Novo Allé, DK 2880 Bagsvaerd, Denmark,
§ Athens University, Biology Department, Division of
Biochemistry and Molecular Biology, Panepistimiopolis, Kouponia, 15701 Athens, Greece, and the ¶ Institute of Biotechnology, Department
of Technical Microbiology, Technical University Hamburg-Harburg,
21071 Hamburg, Germany
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES
ABSTRACT
A gene encoding a highly thermostable
extracellular -amylase from the hyperthermophilic archaeon
Pyrococcus furiosus was identified. The gene was cloned,
sequenced, and expressed in Escherichia coli and
Bacillus subtilis. The gene is 1383 base pairs long and encodes a protein of 461 amino acids. The open reading frame of the
gene was verified by microsequencing of the recombinant purified enzyme. The deduced amino acid sequence is 25 amino acids longer at the
N terminus than that determined by sequencing of the purified protein,
suggesting that a leader sequence is removed during transport of the
enzyme across the membrane. The recombinant -amylase was biochemically characterized and shows an activity optimum at pH 4.5, whereas the optimun temperature for enzymatic activity is close to
100 °C. -Amylase shows sequence homology to the other known
-amylases and belongs to family 13 of glycosyl hydrolases. This
extracellular -amylase is not homologous to the subcellular -amylase previously isolated from the same organism.
INTRODUCTION
One of the most abundantly distributed polysaccharides in nature
is starch, which is produced by plants; it is composed of two high
molecular weight compounds, amylose and amylopectin. Amylose is a
linear chain of glucose residues linked with an -1,4 bond.
Amylopectin is a branched polymer where the -1,4-linked glucose
residues are branched every 17-26 residues with -1,6-linked points.
A wide variety of microorganisms are able to degrade and utilize this
natural high molecular weight biopolymer by secreting starch-degrading
enzymes. These enzymes act either from the nonreducing end of the chain
acting as exo-enzymes producing low molecular weight products
(i.e. -amylase, glucoamylase, and -glucosidase) or in
the interior of the chain and in a random fashion acting as
endo-enzymes and producing linear and branched saccharides with various
lengths (i.e. -amylase).
A great number of -amylases (E.C. 3.2.1.1) have been isolated from a
variety of eucaryotic and procaryotic organisms, and they are described
in a previous reports (1, 2). All of them have been compiled in family
13 of the classification of the glycosyl hydrolase superfamily
described in Ref. 3. Two -amylases from Dictyolglomus
thermophilum (4) and Pyrococcus furiosus (5, 6), could
not be classified in family 13 and have been included in newly
established family 57.
P. furiosus is an anaerobic marine heterotroph growing
optimally at 100 °C and initially was isolated and characterized
(7). Two reports on the identification of -amylase activity from
cell homogenates as well as in the culture medium have been published (8, 9) from this organism. One -amylase, which is a subcellular enzyme, has been purified (5), cloned, and overexpressed in Escherichia coli (6).
In this paper, we present the gene isolation, gene cloning, sequencing,
and expression in E. coli and Bacillus subtilis
and biochemical characterization of a new extracellular -amylase from P. furiosus. Primary structure analysis and comparison
with known -amylase revealed that this enzyme belongs to family 13 of glycosyl hydrolases and does not show sequence homology to its
subcellular counterpart from the same organism.
MATERIALS AND METHODS
Chemicals and Media
Chemical for gel electrophoresis were
from Serva (Heidelberg, Germany); the various substrates were from
Fluka (Buchs, Switzerland). Restriction endonucleases were purchased
from New England Biolabs and Boehringer Mannheim and used as
recommended by the manufacturers. T4 DNA ligase was purchased from New
England Biolabs and used as recommended by the manufacturer. All the
other chemicals were from Merck (Darmstadt, Germany).
The medium for visualization of amylase activity was Luria Broth agar
containing (per 500 ml of agar) 10 ml of a dyed amylopectin solution
prepared as follows: 12.5 g of amylopectin (Serva 2000-4000 kDa)
was dissolved by boiling in 250 ml of water and cooled to room
temperature, 30 ml of 4 M NaOH and 2.5 g of Cibacron
Rot B were added, and the solution was incubated overnight. pH was adjusted to 7 with 4 M HCl. 500 ml of 96% ethanol was
added with stirring to precipitate the amylopectin as a red, viscous
precipitate. The supernatant was discarded, and the amylopectin was
dissolved in 200 ml of water by slight heating. The precipitation with
ethanol was repeated once more. The amylopectin was again dissolved in 200 ml of water and autoclaved and was then ready for use.
Bacterial Strains
E. coli has been described in
Ref. 10, and cells were prepared for and transformed by electroporation
using a Gene PulserTM electroporator from Bio-Rad as described by the
supplier. B. subtilis DN1885 has been described by
Diderichsen et al. (10), and competent cells were prepared
and transformed as described in Ref. 11.
Plasmids
pSJ1678 was used as cloning vector in the
construction of the gene library, and pUC19 (12) was used for
subclonings. The experimental techniques used to construct the plasmids
were standard techniques within the field of recombinant DNA technology
(13). Preparation of plasmid DNA from all strains was constructed by the method described in Ref. 14.
Cloning of the P. furiosus -Amylase Gene
Genomic DNA
from P. furiosus DSM3638 was isolated by the method
described in Ref. 15. Approximately 100 µg of DNA was partially digested with Sau3A and size-fractionated on a sucrose
gradient, and fragments between 3 and 7 kb1
were pooled.
The cloning vector pSJ1678 (see Fig. 1) was digested with
BamHI, and a 3.8-kb fragment was purified from an agarose
gel. Approximately 0.75 µg of vector fragment was ligated to ~4
µg of size-fractionated P. furiosus chromosomal DNA and
used to transform E. coli SJ2 by electroporation.
Fig. 1.
The cloning vector used was pSJ1678.
This plasmid is a shuttle vector that replicates in E. coli
and in Bacillus sp. Upon cloning, the kanD gene
fragment is removed by digestion with BamHI, and the
fragments to be cloned are inserted in its place. PamyM is
the promoter from a Bacillus amylase, which then reads into
the cloned insert from either direction to ensure the expression of the
cloned genes. If nothing is cloned, the two PamyM promoters create an inverted repeat, and plasmids with inverted repeats of this
size are not viable. There is therefore a positive selection for
recombinant plasmids.
[View Larger Version of this Image (28K GIF file)]
The gene bank was plated on LB plates containing dyed amylopectin and
supplemented with 6 µg/ml chloramphenicol. Following overnight
incubation at 37 °C, each plate was replica plated onto two new
plates, which were then incubated overnight at 37 °C. One of these
was subsequently incubated at 60 °C overnight (see Fig. 2).
Fig. 2.
Outline of the methodology applied to
isolate, clone, and detect DNA fragments of chromosomal DNA from
P. furiosus exhibiting amylolytic activity. A short
description of this experimental approach is under "Material and
Methods."
[View Larger Version of this Image (27K GIF file)]
Subcloning of P. furiosus -Amylase Gene
pS2467 was
digested with ClaI, the 4.5-kb fragment containing the
-amylase gene was ligated to AccI-digested pUC19 DNA, and the ligation mixture was transformed into E. coli SJ2.
Transformants were obtained containing the insert in each of the two
possible orientations with respect to the cloning vectors. These clones were SJ2481 containing the pSJ2481 construct and clones SJ2482 containing pSJ2482 construct.
Southern Analysis
pSJ2481 was 32P-labeled by
nick translation using a commercial kit obtained from Amersham Corp.
and used as a probe in a Southern analysis. Hybridization was overnight
at 60 °C in 10 × Denhardt's solution, 1% SDS, 10 mM EDTA and 5 × SSC followed by two 15-min washes in
2 × SSC, 0.1% SDS at room temperature and one 15-min wash at
60 °C (see Fig. 3).
Fig. 3.
The Southern blot analysis of five clones
(lanes 4-8). Four clones (lanes 6-8 and
4) contain a common DNA region around 6.5 kb. That region is
also found in P. furiosus chromosome. The same region (same
HindIII pattern) is found in P. woesei DNA. Experimental details are under "Material and Methods."
[View Larger Version of this Image (38K GIF file)]
DNA Sequencing
4.5 kb of the P. furiosus DNA
insert clones on pSJ2467 was sequenced on both strands, using
SequenaseTM (16) and a combination of subclones and oligonucleotide
primers based on previously determined sequences.
Expression of the -Amylase Gene in B. subtilis
The
plasmid pSJ1678 used for construction of the gene library is a shuttle
vector able to replicate both in E. coli and B. subtilis. To test for expression of the amylase activity in
B. subtilis, pSJ2467 was therefore transformed into
competent cells of DN1885 selecting for resistance to chloramphenicol
(6 µg/ml) on LB plates containing dyed amylopectin. 10 transformants
were picked onto two new plates with dyed amylopectin along with
SJ1678, which is DN1885/pSJ1678 as a control. After incubation
overnight at 37 °C, one plate was transferred to 65 °C, whereas
the other was kept at 37 °C. Seven hours later, the degradation of
the amylopectin around the 10 transformants with pSJ2467 was apparent
on the plate incubated at 65 °C because of the formation of a clear
halo. No degradation halo was formed around the control strain (see
Fig. 6).
Fig. 6.
The amylase was expressed in B. subtilis by transforming the recombinant clone pSJ2467 obtained
from the screening of the gene bank in E. coli into
B. subtilis. Ten tranformants and the control at 37 and 60 °C were plated.
[View Larger Version of this Image (39K GIF file)]
Analytical Methods
Preparative polyacrylamide gel
electrophoresis was performed in 1.5-mm-thick polyacrylamide gels
(either homogeneous (5 or 12%, w/v) or gradient gels (5-30%, w/v))
at a constant voltage of 400 V for 24 h at 4 °C. The protein
bands were visualized by silver staining (17). Analytical 11.5%
polyacrylamide slab electrophoresis in the presence 0.1% SDS was
carried out at a constant current of 40 mA/gel for 3 h.
Commercially available molecular weight markers were used to calibrate
the gel. The protein band exhibiting -amylase activity on the gel
was detected by soaking the gels in 50 mM acetate buffer,
pH 5.5, containing 1% (w/v) starch for 1 h at 4 °C, further
incubating the gels at 90 °C for 30 min, and staining the gels with
0.15% (w/v) iodine and 1.5% (w/v) potassium iodide until a clear zone
became visible.
Amylase activity was determined in the cell supernatant as described in
Ref. 18. In a typical assay, enzyme solution up to 100 µl was added
to 250 µl of sodium acetate buffer (50 mM, pH 5.5)
containing 1% (w/v) starch and incubated at 95 °C for 30 and 60 min. One unit of -amylase is defined as the amount of the enzyme
that liberates 1 µmol of reducing sugar/min with maltose as a
standard.
The activity- and temperature-dependent experiments were
carried out in a water bath for the range between 40 and 90 °C,
whereas for the range between 90 and 130 °C a glycerol bath was
used. Activity tests above 100 °C were carried out in closed Hungate tubes to prevent boiling of the solution. For the determination of pH
optimum activity, the following buffers were used for the different pH
ranges: for pH 3.5-4.0, 50 mM citrate; for pH 4.5-6.0, 50 mM sodium acetate; and for pH 6.5-7.0, 50 mM potassium phosphate.
The substrate specificity of the -amylase was studied in 50 mM sodium acetate at pH 5.5 using 0.5 unit of purified
enzyme/ml of reaction at 90 °C for 30 min. The final concentration
of various substrates was 1% (w/v). Thermal stability experiments were
performed at the indicated temperatures in the above mentioned baths,
and the residual activity was determined in a typical enzyme assay solution.
Sugars released by the enzymatic action of -amylase from P. furiosus on starch were analyzed as follows. For each milliliter of acetate buffer (50 mM, pH 5.0), 0.5 unit of -amylase
was added. The final starch concentration was 1% (w/v), and the
incubation was conducted at 90 °C. Samples were withdrawn after
various time intervals. Each sample was purified with ion exchange
resin (Serdolyt MB, Serva, Heidelberg, Germany), and the sugars were
analyzed by HPLC using an Aminex HPX-42 A column (Bio-Rad, Richmond,
CA). Sugars eluted were monitored by a differential refractometer
(Knauer, Bad Homburg, Germany).
Computer Analysis
The search for sequence homology to the
other amylases performed at the National Center for Biotechnology
Information using the BLASTP network service (19).
RESULTS
Cultivation of P. furiosus
P. furiosus (DSM 3638)
was cultivated at 98 °C in a medium as described in Ref. 20.
20-liter cultures were continuously gassed with
H2/CO2 (80:20). Cell growth was paralleled by
enzyme production and degradation of starch in the medium. By using
continuous gassing, an enzyme activity above 1000 units/liter was
detected. These growth conditions caused about 80% secretion of the
enzyme into the culture fluid. The amount of the secreted enzyme
reached the maximum after 18 h of cultivation (data not
shown).
Cloning and Sequencing of the -Amylase Gene of P. furiosus
The preparation of the DNA expression library was
carried out as described under "Materials and Methods." Fig.
1 shows the pSJ1678 vector that was used to clone and
express the P. furiosus DNA library.
Fig. 2 outlines the clone selection procedure used to
isolate the -amylase gene, which is based on the secretion of
amylolytic activity from the recombinant cells and the detection of
this activity around the colony(ies) by producing a clear halo. The qualitative detection of amylase activity on agar plates is also described under "Materials and Methods." Among 10,000 colonies, five colonies have shown clear halos indicating degradation of the
amylopectin on the 60 °C agar plates, whereas no halos were observed
around the colonies on the plates that were kept at 37 °C. These
five clones were taken from the 37 °C plates and named SJ2463 through SJ2467.
Restriction digests using HindIII revealed that the P. furiosus DNA inserts on the four clones SJ2463, SJ2464, SJ2465,
and SJ2467 shared a common DNA region without the inserts being totally identical, whereas the DNA contained on SJ2466 clone appeared unrelated
to the other four clones. We used the construct pSJ2467, which
contained an insert of approximately 4.5 kb, for further analysis.
pSJ2467 was digested with ClaI, the 4.5-kb fragment
containing the -amylase gene was ligated to AccI-digested
pUC19 DNA, and the ligation mixture was transformed into E. coli SJ2. Transformants were obtained containing the insert in
each of the two possible orientations with respect to the cloning
vector. These were SJ2481 containing pSJ2481 and SJ2482 containing
pSJ2482 (data not shown). Both clones produce -amylase as detected
on amylopectin plates at 60 °C. Fig. 4 schematically shows the
various subconstructs and their phenotype, which represents the
-amylase activity.
Fig. 4.
Restriction map of the cloned DNA; a number
of subclones are indicated. The phenotype of the clones plated on
dyed amylopectin plates shows the -amylase activity. At the
bottom, the location of the -amylase encoding gene as
deduced from the DNA sequence is indicated.
[View Larger Version of this Image (15K GIF file)]
Further subcloning was preformed from pSJ2481. pSJ2487 (see Fig. 4) was
constructed by deletion of the 1-kb XbaI fragment from
pSJ2481 and transformation of the religated plasmid into E. coli SJ2. The resulting transformants were not able to produce halos on LB plates containing dyed amylopectin, indicating that this
deletion had removed a DNA region of importance for expression of an
active amylase protein. The 1-kb XbaI fragment from pSJ2481 was inserted into XbaI-digested pUC19 to give pSJ2489 and
pSJ2490 (identical), which were used for sequencing.
When a Southern blot (Fig. 3) prepared with digested
genomic DNA from P. furiosus was probed with the
32P-labeled pSJ2481, a 5.3-kb PstI fragment, a
3.1-kb HindIII fragment, a 5.3-kb XhoI fragment,
and two EcoRI fragments of 0.7 and 2.4 kb were found to
specifically hybridize to the probe. The blot also shows that pSJ2463,
pSJ2464, pSJ2465, and pSJ2467 contain a common DNA region (a
HindIII fragment of approximately 0.5 kb is common to
pSJ2463, pSJ2464, pSJ2465, pSJ2467, and chromosomal P. furiosus DNA). It also proves that the insert of pSJ2481 is derived from the chromosome of P. furiosus and that a
homologous DNA region exists in the chromosome of Pyrococcus
woesei. The chromosomal P. woesei DNA was isolated
according to the method described in Ref. 15. Thus pSJ2481 hybridizes
to exactly the same fragments in HindIII-digested P. woesei DNA as in HindIII-digested P. furiosus DNA. Both clones produced -amylase as visualized by
the appearence of clear halos on dyed amylopectin plates after incubation at 60 °C. The amylase-producing transformants look different when compared with transformants containing the pUC19 vector
plasmid only. They form smaller and more translucent colonies.
The open reading frame corresponding to the -amylase gene was
localized by subcloning (the ability of individual subclones to produce
-amylase was assayed on plates containing dyed amylopectin) as
outlined on Fig. 4. The 4.5 kb of the P. furiosus DNA insert cloned on pSJ2467 was sequenced on both
strands using SequenaseTM and a combination of subclones and primer
walking.
The DNA sequence of the -amylase coding region, including the signal
peptide coding region, is shown in Fig. 5. On the basis of the DNA sequence and N terminus amino acid sequence determination of
the mature -amylase, the amino acid sequences of the signal peptide
and of the mature -amylase have been deduced. The signal peptide is
25 amino acids long and is cleaved between Ala25 and
Ala26.
Fig. 5.
Nucleotide sequence and deduced amino acid
sequence of the -amylase gene from P. furiosus. The
nucleotide sequence between the 228 and 1512 sites is presented. The
ribosome binding site GGAGGT ( 6 to 9) 6 bases upstream of the start
codon GTG (+1) is in italics. The promoter region containing
a consensus BoxA sequence TTTATA ( 53 to 58) is
underlined. The site of the cleavage of signal peptide is
between Ala25 and Ala26 and indicated by a
dot. Immediately following the TGA stop codon there is a
pyrimidine-rich sequence (which is between 1383 and 1401 and
underlined) as found in other Pyrococcus sp. and
archaeal genes. The -amylase gene has been deposited in the
GenBankTM under the accession number [GenBank].
[View Larger Version of this Image (51K GIF file)]
The DNA fragment containing the -amylase gene encompasses 1740 nucleotides, with the initiation codon GTG at position +1 (Fig. 5). The
1380-base pair open reading frame encodes a single polypeptide with a
molecular mass of 52,843 Da. This agrees well with the apparent
molecular mass of the protein, determined by gel electrophoresis under
denaturing conditions, of 54 kDa. Immediately upstream of the coding
region is the sequence GAGGT identical to the putative ribosome-binding
site of the glyceraldehyde-3-phosphate dehydrogenase gene of P. woesei (GAGGT) (21). A pyrimidine-rich region exists immediately
downstream from the TAG termination codon in the -amylase gene, like
other archaebacterial sequences. A "box A" promoter region was also
identified between 53 and 58. The G + C content of the -amylase
gene is 42.9%, slightly higher than the value reported for the total
genome of 38% (7). As has been seen in other sequenced genes from
extreme thermophiles, A and T are preferred bases in the third position
of the codon (21).
Expression of the -Amylase Gene in E. coli and B. subtilis
The plasmid pSJ1678 used for construction of the gene
library is a shuttle vector able to replicate in both E. coli and B. subtilis. The amyM promoters,
reading into the inserts cloned in this vector, are functional in
E. coli, thus enhancing the chances that any gene cloned
would be successfully expressed in this host. To examine the expression
of the amylase activity in B. subtilis, pSJ2467 was
therefore transformed into competent cells of DN1885 selecting for
resistance to chloramphenicol (6 µg/ml) on LB plates containing dyed
amylopectin. Few transformants were picked onto new plates with dyed
amylopectin, and after incubation at 37 °C one plate was transferred
to 65 °C, whereas the other was kept at 37 °C. Seven hours later
a degradation of the amylopectin around the transformants with pSJ2467
was observed on the plates incubated at 65 °C as formation of a
clear halo. No halo was formed at 37 °C for the control strain as
shown in Fig. 6.
Biochemical Characterization
As shown in Fig.
7 (A-C), -amylase from P. furiosus is active in a broad temperature range from 40 to
130 °C (Fig. 7B) and in a pH range from 3.5 to 8.0 (Fig.
7A) Maximal activity is measured at 100-105 °C and pH
4.5. Conditions for incubation of P. furiosus -amylase
for the determination of pH and temperature optimum are described under
"Materials and Methods."
Fig. 7.
A, activity pH dependency of -amylase
from P. furiosus. The extract containing the enzyme was
incubated in water baths for measurements up to 90 °C or in glycerin
bath for measurements between 90 and 130 °C. After various time
intervals, as indicated on the plot, samples were withdrawn and
analyzed for -amylase activity as described under "Materials and
Methods." B, temperature-dependent recombinant
P. furiosus -amylase activity. The reaction was carried out at the standard assay as described under "Materials and
Methods" and at various temperatures as indicated on the plot.
C, thermal stability of -amylase from P. furiosus. The samples containing the enzyme were incubated at
various temperatures (as indicated on the plot), and after various time
intervals samples were withdrawn and the -amylase activity was
determined as described under "Materials and Methods."
[View Larger Version of this Image (13K GIF file)]
Beside the extremely high temperature optimum, the -amylase shows
remarkable thermal stability as well as stability against chemical
denaturation. As depicted in Fig. 7C, incubation in a boiling water bath for 6 h causes a decrease in enzymatic activity of only 20%. Around 60% of the enzyme activity is still detectable after 120 °C for 1 h. Furthermore, after heating of the enzyme at 115 °C for 3 h, 35% of residual activity was determined.
The same sample was able to recover about 75% of its initial activity after 3 h of incubation (renaturation period) at room temperature. Some activity is still detectable after 30 min at 130 °C. On the other hand, the -amylase is 55% active in the presence of 1.5 M urea or 61% in 0.3 M guanidine
hydrochloride. The initial activity can be completely recovered after
removing both denaturing agents by dialysis.
The addition of 5 mM of molybdenum, calcium, or magnesium
ions did not influence -amylase activity. A slight decrease of activity could be detected in the presence of cobalt, nickel, and iron
ions, and complete inhibition was found when 5 mM of zinc
or copper ions was added. Because EDTA did not show any effect, we can
assume that the addition of metal ions is not required for enzymatic
activity (data not shown).
Substrate Specificity
The partially purified extracellular
-amylase from P. furiosus hydrolyzes native starch,
soluble starch, amylopectin, maltodextrin, and amylose as shown in
Table I. Main products of starch degradation were
oligosaccharides such as maltohexaose, maltopentaose, maltotetraose, maltotriose, and maltose. The enzyme degrades the -1,4 glycosidic linkage in starch in a random fashion and can be designated as an
-amylase.
HPLC analysis has shown that the distribution of oligosaccharides seen
in the chromatograms is typical of endo-amylase attack. The major
oligosaccharides formed on prolonged hydrolysis are DP4, DP6, and DP7
(data not shown).
Comparison of -Amylase from P. furiosus to the Other Known
-Amylases
The National Center for Biotechnology Information
BLAST e-mail server was used to search the peptide sequence data bases
(Brookhaven Protein Data Bank, SwissProt, Pacific Investment Research,
Inc. and GenPept) for proteins homologous to the -amylase protein sequence (19, 22). The -amylase encoded by this open reading frame
revealed homology to -amylases and other starch-degrading enzymes
from a variety of organisms including bacteria, insects, and plants,
and it is classified in family 13 of the glycosyl hydrolases (3).
Nakajima and his colleagues (23) have identified four short primary
sequence motifs, which also have been identified in amylolytic enzymes
with other activities. These motives were also found in our -amylase
and indicated as regions I to IV in the alignment shown in Fig.
8A.
Fig. 8.
A, alignment of the conserved regions
(I-IV) between our -amylase (Amy1_Pyrfu) and the other four that
have shown the highest homology scoring. B, table indicating
the percentage of homology (identity) between our -amylase and the
other four highly related enzymes described under "Results."
[View Larger Version of this Image (18K GIF file)]
The highest scoring homologous sequences were all -amylases, of both
procaryotic and eucaryotic origin. A homology matrix based on the
alignment is presented in Fig. 8B. It reveals that the
P. furiosus -amylase has about 47% homology to
Bacillus licheniformis (Amy-Bacli) (24), 44% to
Salmonella typhimurium (Amy2-Salty) (25), 39% to
Oryza sativa (Amy1-Oryza) (26), and 34% to the Pseudomonas stutzeri (Amt4-Psest) (27) enzymes.
Comparison of the Subcellular and Extracellular -Amylases from
P. furiosus
Laderman et al. (5, 6) have also isolated
and characterized a subcellular -amylase from P. furiosus. We named our -amylase "extracellular enzyme" to
distinguish it from the subcellular enzyme. There are several
differences between the two enzymes in terms of size, localization, and
primary structure, which are summarized in Table II. Our
extracellular -amylase shows significant homology to the other
-amylase from a variety of organisms and has been classified in
family 13 of glycosyl hydrolases, whereas the subcellular -amylase
shows strong homology only to -amylase from D. thermophilum, and both have been included to an extra family 57 of
glycosyl hydrolases.
DISCUSSION
We have isolated and sequenced the gene of a new extracellular
-amylase from the hyperthermophile archaeon P. furiosus.
The gene was also expressed in E. coli and B. subtilis using a novel shuttle vector. The structure of the gene
displays the typical characteristics of an archaeon gene with a typical
ribosome binding site and a pyrimidine-rich region immediately
downstream from the stop codon. The utilization of the GTG initiation
codon, which is used relatively rarely, seems to be the rule in
Pyrococcus sp. genes isolated so far.
The extracellular -amylase enzyme is not very closely related to any
other amylases of family 13 of glycosyl hydrolases. On the other hand
it can be aligned to the other enzymes, and it has the conserved
regions I-IV found in other amylases.
From the structural features of the extracellular -amylase gene and
the subcellular described by Laderman et al. (5, 6) as well
as their primary structure comparison, it is clear that these are two
completely different enzymes. This archaeon converts starch or glycogen
to small linear and branched oligosaccharides, which can be transported
most probably by "dextrin premease" into the cell. The presence of
intracellular -amylase indicates that further carbohydrate
metabolism by P. furiosus is performed intracellularly.
The extracellular -amylase from P. furiosus is one of a
number of extremophilic enzymes that have been expressed in a
mesophilic host in an active form. The fact that expression of amylase
activity from a Pyrococcus -amylase gene can be obtained
in B. subtilis without any modification of the gene (for
example replacement of the ribosome binding site) to allow more
efficient initiation of translation is surprising. B. subtilis is quite restrictive in its acceptance of ribosome
binding sites, and it is a frequent observation that cloned genes from
non-Gram-positive organisms would not be expressed in B. subtilis without proper modification of their expression signals.
To our knowledge, this expression of the P. furiosus
-amylase constitutes the first example of expression from an
unmodified (or nonengineered) Pyrococcus gene in
Bacillus.
The high thermostability of this pyrococcal -amylase, its
independence on metal ions, its unique substrate specificity, and its
product pattern make this enzyme an interesting candidate for
industrial application. It is therefore very important to employ
genetic and fermentation techniques for the production of such enzymes
on a large scale.
FOOTNOTES
*
This work was supported by the European Union, Project
Biotechnology of Extremophiles, Contract BIO-CT93-02734, and by the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U96622[GenBank].
To whom correspondence should be addressed: Inst. of
Biotechnology, Dept. of Technical Microbiology, Technical University Hamburg-Harburg, Denickestr. 15, 21071 Hamburg, Germany. Tel.: 49-40-7718-3117; Fax: 49-40-7718-2909.
1
The abbreviations used are: kb, kilobase(s);
HPLC, high pressure liquid chromatography.
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©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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