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Journal of Bacteriology, March 1999, p. 1409-1414, Vol. 181, No. 5
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Cloning and Expression of the algL Gene,
Encoding the Azotobacter chroococcum Alginate Lyase:
Purification and Characterization of the Enzyme
Ana
Peciña,
Alberto
Pascual, and
Antonio
Paneque*
Instituto de Bioquímica Vegetal y
Fotosíntesis, Consejo Superior de Investigaciones
Científicas-Universidad de Sevilla, Seville, Spain
Received 30 July 1998/Accepted 15 December 1998
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ABSTRACT |
The alginate lyase-encoding gene (algL) of
Azotobacter chroococcum was localized to a 3.1-kb
EcoRI DNA fragment that revealed an open reading frame of
1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in
agreement with the value previously reported by us for this protein.
The deduced protein has a potential N-terminal signal peptide that is
consistent with its proposed periplasmic location. The analysis of the
deduced amino acid sequence indicated that the gene sequence has a high
homology (90% identity) to the Azotobacter vinelandii gene
sequence, which has very recently been deposited in the GenBank
database, and that it has 64% identity to the Pseudomonas
aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in
other bacteria. The A. chroococcum AlgL protein was
overproduced in Escherichia coli and purified to
electrophoretic homogeneity in a two-step chromatography procedure on
hydroxyapatite and phenyl-Sepharose. The kinetic and molecular
parameters of the recombinant alginate lyase are similar to those found
for the native enzyme.
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INTRODUCTION |
Alginates are linear polysaccharides
composed of (1,4)-linked 
-D-mannuronic acid and its C-5
epimer, 
-L-guluronic acid. These uronic acids are
arranged in block structures which may be homopolymeric (polymannuronic
acid or polyguluronic acid) or heteropolymeric random sequence
(15). The proportion and arrangement of the block structures
vary greatly in alginates from different sources and determine the
physical properties of the polymer, particularly the ability to form
gels in the presence of divalent cations (21). Alginates are
synthesized as cell wall components by brown seaweeds and as
exopolysaccharides by two families of heterotrophic bacteria,
Pseudomonadaceae and Azotobacteriaceae. Marine
alga alginate is used widely in the food, pharmaceutical, textile, and
oil industries. Bacterial alginate is an acetylated polymer of
D-mannuronic and L-guluronic acids, and
evidence has been presented for the location of O-acetyl
groups at the 2 and/or 3 position of D-mannuronosyl
residues (41). In their pioneering work, Lawson and Stacey
(26) described the existence of two capsular polysaccharides
in Azotobacter chroococcum, and, more recently, one of these
exocellular polysaccharides was identified as an alginate
(9). In cystic fibrosis patients, Pseudomonas aeruginosa produces alginate, which facilitates the attachment of
the bacterium to tracheal mucins. The exopolysaccharide protects the
microorganism from phagocytes and prevents antibiotic uptake. Consequently, it is a major pathogenic factor in these patients (3).
Alginates are degraded by a group of enzymes that catalyze the
-elimination of the 4-O-linked glycosidic bond, with formation of
unsaturated uronic acid-containing oligosaccharides (6). Several of the bacteria that synthesize alginate-like polysaccharides also produce alginate lyases, but they cannot use the polymers as the
sole carbon and energy source. Typically, alginate lyases have an
absolute specificity for either D-mannuronic or
L-guluronic acid at the nonreducing side of the bond to be
cleaved but no limitation on the uronic acids at the reducing side
(5). Although alginate lyase may prove useful in the
elucidation of alginate molecular structure, its degrading action on
alginate represents a drawback in the bioproduction of the polymer by
fermentation. In studies of P. aeruginosa, it is necessary
to look for inhibitors of alginate synthesis with potential use as
therapeutic agents in cystic fibrosis patients. Therefore, research on
this enzyme is important from different points of view.
In this paper, we describe the cloning of the algL gene
encoding the alginate lyase from A. chroococcum and the
purification and characterization of its product. We also report the
conditions under which the enzyme can be overproduced in
Escherichia coli and easily purified to electrophoretic homogeneity.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and culture conditions.
A.
chroococcum ATCC 4412 was grown at 30°C on nitrogen-free Burk's
medium supplemented with 0.5% (wt/vol) sucrose as the sole energy and
carbon source.
E. coli DH5 (Bethesda Research Laboratories) was used for
all plasmid constructions, E. coli MC1061 (30)
was used for gene library construction, and E. coli BL-21
(DE3) (40) was used as the host for pAPET-2 to produce
alginate lyase. These strains were grown on Luria-Bertani (LB) and M9
minimal media containing ampicillin (100 µg/ml) as described by
Sambrook et al. (37).
P. aeruginosa 8830, a gift from A. M. Chakrabarty
(University of Illinois, Chicago), was grown as described by May and
Chakrabarty
(
29) to isolate alginic acid, which was used as
substrate for
A. chroococcum alginate
lyase.
Plasmid pRL500 (
12) was used to construct the partial
A. chroococcum gene library, and pBluescript II SK(+)
(Stratagene)
was used as cloning vector. Plasmid pET-3a, used for the
expression
of recombinant proteins, was from Novagen. pCL8, containing
alginate
lyase gene (
algL) from
P. aeruginosa
(
4), was a gift from A.
M. Chakrabarty. Plasmids
pAPL4.7 and pAPET-2, containing the
algL gene from
A. chroococcum, were constructed for this
work.
DNA manipulation and Southern blot hybridization.
Total DNA
from A. chroococcum was isolated as described by Ausubel et
al. (1). All DNA manipulations and E. coli
transformations were performed by standard procedures (37).
DNA fragments were purified from agarose gels by using the GeneClean
kit (Bio 101, Inc.). For Southern hybridization, DNA was digested and
fragments were electrophoresed on 0.7% agarose gels with the
Tris-borate-EDTA buffer system (37). DNA was transferred to
Z-probe membranes (Bio-Rad) under vacuum, and Southern blot
hybridizations were performed as described by Ausubel et al.
(1). DNA probes were 32P labelled with a
DNA-labelling kit (-dCTP) (Pharmacia) and [-32P]dCTP (Amersham).
Cloning of the algL gene.
Genomic DNA from
A. chroococuum was completely digested with EcoRI
and fractionated on a 0.7% agarose gel. DNA fragments of approximately
2.0 to 4.0 kb were purified and ligated into the EcoRI site
of pRL500. E. coli MC1061 was transformed with the ligation
mixture, and transformants were grown on LB agar plates containing
ampicillin. Transformants were screened for the presence of the
algL gene by colony hybridization blotting with a 1.8-kb XbaI-EcoRI fragment from plasmid pCL8 as a probe.
DNA sequencing and nucleotide sequence analysis.
DNA
fragments containing the algL gene were subcloned into
pBluescript II SK(+) and sequenced by the dideoxy chain termination method (38) with M13 universal and reverse oligonucleotides or synthetic oligonucleotides as primers. Sequencing reactions were
carried out with Sequenase version 2.0 (US Biochemical Corp.). Nested
unidirectional deletions were generated with the double-stranded nested-deletion kit (Pharmacia LKB). Both strands of DNA were sequenced.
Computer sequence analysis was carried out with the Genetics Computer
Group software package (
10). Amino acid sequences
were
compared with the FASTA program, and alignments were produced
with the
PileUp program and default parameters (
33).
Expression of AlgL and purification of alginate lyase.
To
express AlgL protein, a PCR with plasmid template pAPL4.7 and primers
designed to introduce the NdeI
(5'-TATTCATATGAAGACCAGACTTGCCC-3') and BamHI
(5'-CTTCGGGATCCCTGCGGAATACCAG-3') restriction sites encompassing algL was performed by using a final volume of
50 µl that contained 1.5 ng of DNA from plasmid pAPL4.7, 0.2 mM each deoxynucleoside triphosphate, 50 pmol of each oligonucleotide, and 2.5 U of Taq polymerase and 1× buffer (Boehringer).
An expected PCR product of 0.2 kb containing
BamHI and
NdeI sites was amplified. It was digested with
NdeI and
BamHI, ethanol
precipitated, and ligated
into a similarly digested and phosphatase-treated
pET-3a expression
plasmid. Possible constructs were confirmed
by restriction digestion
and
sequencing.
To create pAPET-2, a 1.8-kb
BstXI-
EcoRV fragment
from pAPL4.7 (containing the
algL gene) was isolated and
cloned in pET-3a
(containing the PCR product) previously digested with
BamHI, refilled
by a Klenow reaction, and finally digested
with
BstXI. This plasmid
contained the whole
A. chroococcum algL gene under the control
of the T7/
lac
promoter; in consequence, its expression was inducible
by
isopropyl-
-
D-thiogalactopyranoside
(IPTG).
E. coli BL-21 (DE3) cells were transformed with plasmid
pAPET-2. Transformation mixtures were used to directly inoculate 2
liters of M9 minimal medium containing 100 µg of ampicillin per
ml.
The culture was incubated at 30°C to an absorbance at 550
nm
(
A550) of 0.5; at this time, T7 polymerase was
induced by adding
IPTG at a final concentration of 1 mM, and cells were
incubated
for another 3 h at 30°C. Then cells were collected by
centrifugation
(8,000 ×
g for 5 min), and the
periplasmic fraction was prepared
as described by Efterkhar and
Schiller (
11). The periplasmic
proteins were loaded onto a
hydroxyapatite column (1.8 by 14 cm)
equilibrated with 20 mM potassium
phosphate buffer (pH 7.5). The
adsorbed alginate lyase activity was
eluted with a linear gradient
of 0.1 to 1.0 M potassium phosphate
buffer (pH 7.5). Most active
fractions were pooled and loaded onto a
phenyl-Sepharose column
(1 by 8 cm) equilibrated with 25 mM Tris-HCl
buffer (pH 8.2) containing
1 M NaCl. The alginate lyase activity was
eluted within the void
volume of this column. Most active fractions
were combined and
used as the purified AlgL
enzyme.
Alginate lyase assay.
As the substrate for the enzyme assay,
alginic acid from either Macrocystis pyrifera (60%
mannuronate; Sigma), A. chroococcum, or P. aeruginosa was used. Alginic acid from A. chroococcum
was prepared as described by Jarman et al. (22), and that
from P. aeruginosa was prepared as described by May and
Chakrabarty (29). When needed, the substrate from P. aeruginosa was deacetylated before being used as substrate
(16). Alginate lyase activity was quantitatively measured by
the thiobarbituric acid method (42). Enzyme preparations (5 to 15 µg of total protein) in 50 mM Tris-HCl buffer (pH 7.5)-0.2 M
MgCl2 containing substrate (0.1 mg) were incubated at
30°C for 15 min. One unit of enzyme activity is defined as the amount
of enzyme required to generate 1 µmol of -formylpyruvate per min.
The protein concentration was measured by the method of Bradford
(8) with bovine serum albumin (Sigma) as the standard.
Alginate lyase plate assay.
Alginate lyase-producing
E. coli strains were identified by being grown at 37°C on
LB plates containing 1% agarose, alginate from the seaweed M. pyrifera (2 mg/ml), and 100 µg of ampicillin per ml. The plates
were stained by flooding with 10% (wt/vol) cetylpyridinium chloride,
and clear zones of depolymerization on a white background were
observed, indicating lyase activity (17).
SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis was done by the method of Laemmli
(24). Enzymatic activity after SDS-PAGE and subsequent
renaturation were visualized exactly as described previously
(34). All PAGE runs were performed at room temperature with
a Bio-Rad mini Protean slab gel apparatus.
Nucleotide sequence accession number.
The nucleotide
sequence of the A. chroococcum algL gene (see Fig. 3) has
been deposited in the DDBJ, EMBL, and GenBank DNA databases under
accession no. AJ223605.
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RESULTS AND Discussion |
Cloning of the A. chroococcum algL gene.
To clone
algL, total genomic DNA from A. chroococcum was
digested and subjected to Southern blotting with, as the probe, a 1.8-kb XbaI-EcoRI fragment from plasmid pCL8,
containing the algL gene from P. aeruginosa
(4). A 3.2-kb EcoRI fragment hybridized with this
probe (data not shown). Total A. chroococcum DNA was then
digested with EcoRI, and fragments of approximately 2.0 to 4.0 kb were purified and ligated to pRL500 to construct a partial A. chroococcum gene library. Transformants in E. coli MC1061 were screened for the presence of the algL
gene by using the same probe (see Materials and Methods). A positive
clone was analyzed. This clone contained a 3.1-kb EcoRI
insert, which was cloned into pBluescript to generate plasmid pAPL4.7
for further analysis (Fig. 1).
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FIG. 1.
Restriction map of pAPL4.7 and pAPET-2 containing the
A. chroococcum algL gene. Thin lines and open bars
correspond to vector plasmid DNA and cloned DNA fragments,
respectively. The coding region is represented by solid bars. A broken
arrow indicates the direction of the transcription of algL.
E, EcoRI; A, ApaI; B, BstXI, S,
SacI; N, NdeI; EV, EcoRV. Potential
algX and algI genes upstream and downstream of
algL gene are indicated by solid arrow underlines.
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To determine whether this DNA fragment encoded the alginate lyase
enzyme,
E. coli DH5
cells were transformed with pAPL4.7
and tested for alginate lyase activity on LB plates containing
alginate. A clear zone of depolymerization was observed around
cells
containing pAPL4.7. Untransformed
E. coli DH5
did not
synthesize
an endogenous activity that was able to degrade alginate,
and
DH5
cells containing the pBluescript vector showed no clear
zones
(Fig.
2). Therefore, the 3.1-kb
EcoRI fragment harbors the alginate
lyase enzyme from
A. chroococcum.
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FIG. 2.
Plate assay for alginate lyase activity. Cells were
restreaked on LB plates containing alginate and assayed for alginate
lyase activity (see Materials and Methods). 1, untransformed E. coli DH5 cells; 2, E. coli DH5 cells containing
pAPL4.7 plasmid; 3, E. coli cells containing pBluescript
SK(+) plasmid.
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Nucleotide sequence of the algL gene.
To determine
if the 3.1-kb fragment contains the bonafide algL gene from
A. chroococcum, nested deletions were generated from pAPL4.7
with exonuclease III and a number of the resulting clones were checked
for alginate lyase activity to locate the algL gene in the
pAPL4.7 plasmid and were used for sequencing. The complete nucleotide
sequence of algL gene and its translation of the open reading frame into a 372-residue amino acid sequence are shown in Fig.
3. The coding region ends with a TAG stop
codon and it encodes a polypeptide with a calculated molecular mass of
42.98 kDa. As reported previously, the molecular mass of the alginate lyase from A. chroococcum, determined by activity staining
after SDS-PAGE and subsequent renaturation, is 43 kDa (34)
and the molecular mass estimated by gel filtration is about 46 kDa
(35). A potential Shine-Dalgarno (GAGG) sequence lies just 7 nucleotides upstream from the start site. In the region of DNA upstream
of the ATG codon, sequences related to the 
35 and 10 consensus promoter regions were not identified. The G+C content of the nucleotide sequence is 67.2%, in good agreement with the value found for the
Azotobacter genes, which is within 65 to 68%
(2).
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FIG. 3.
Nucleotide sequence of A. chroococcum algL
and deduced amino acid sequence of its product. The deduced amino acid
sequence of AlgL is shown in single-letter code below the nucleotide
sequence of algL. The possible Shine-Dalgarno sequence is
indicated by a dotted underline. The initial methionine is indicated by
an asterisk, and the possible signal peptide is indicated by a solid
underline.
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When sequences contained in plasmid pAPL4.7 flanking the
algL open reading frame were sequenced and compared with
other nucleotide
sequences deposited in the data banks, it was observed
that these
sequences showed homologies to those of
P. aeruginosa
algX and
algI genes, which are constituents of the
alginate biosynthesis
operon (
14,
31). These data suggest
that the
A. chroococcum algL gene is probably located in an
operon and has, as flanking
genes, the
algX gene at its 5'
end and the
algI gene at its 3'
end. The existence of an
alginate biosynthesis operon has also
been described for
A. vinelandii (
13). The sequence of the
algXLIVFA operon from
A. vinelandii (as appeared
in the GenBank database)
indicates that the alginate biosynthesis
operon in this organism
has the same structure as the one described in
the present report
for
A. chroococcum. The flanking
sequences of the
A. chroococcum algL gene show a high
homology to the recently reported sequence
of the
algXLIVFA
operon from
A. vinelandii.
Deduced amino acid sequence of AlgL.
The deduced protein
sequence of AlgL was compared with entries in the GenBank and
SWISS-PROT databases. Multiple alignment of several alginate lyase
sequences indicates that A. chroococcum AlgL is more similar
to P. aeruginosa AlgL (63.6% identity) than to K. pneumoniae AlgL (16.3% identity), P. alginovora 
lgL
(16.3% identity), or Photobacterium lgL (18.8%). As
mentioned above, the A. vinelandii AlgL deduced amino acid
sequence (GenBank) is very similar (90% identity) to that from
A. chroococcum.
The presence at the N-terminal region of a signal peptide of 20 to 30 amino acids, rich in hydrophobic residues, had been
described for
several alginate lyases (
4,
27,
28,
39).
This signal peptide
has not been experimentally investigated in
A. chroococcum,
but the hydrophobicity profile deduced from the
algL gene
shows a highly hydrophobic region of 25 amino acids
at the
amino-terminal end. A potential cleavage site appears between
amino
acid residues Ala
22 and Ala
23. This zone has
high homology to the amino acid sequence of the
signal peptide
described for
P. aeruginosa. These data suggest
the
existence of a signal peptide in
A. chroococcum alginate
lyase
and the maturing of this protein. The mature protein would then
begin at Ala
23. Processing and export through the inner
membrane are consistent
with the periplasmic location of AlgL in
A. chroococcum (
35)
and its purification from the
periplasmic fraction from
E. coli.
Purification and characterization of recombinant alginate
lyase.
The algL gene was expressed from the
bacteriophage T7 polymerase promoter of pET-3a expression vector. The
final construct, called pAPET-2, was transformed in E. coli
BL21 (DE3), and the AlgL enzyme was produced following induction of T7
polymerase. The best conditions for the overexpression of the enzyme
were direct culture inoculation from the transformation mixture,
30°C, initiation of induction by adding 1 mM IPTG at an
A550 of 0.5, and an induction time of 3 h.
These conditions prevented the overexpressed protein from aggregation
into inclusion bodies.
The gene product of
algL was purified 10.4-fold from the
periplasm fraction of
E. coli BL21 (DE3)(pAPET-2), with a
recovery
of 6% by hydroxyapatite chromatography and phenyl-Sepharose
column
chromatography (Table
1). The
final preparation gave a single
band on SDS-PAGE with a molecular mass
of approximately 43 kDa
(Fig.
4). This
value is in good agreement with the value estimated
from the deduced
amino acid sequence of AlgL and with the value
determined by activity
staining in SDS-PAGE for the native AlgL
from
A. chroococcum
(
34).
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FIG. 4.
SDS-PAGE analysis of the recombinant A. chroococcum AlgL protein. (A) Gel stained with Coomassie brilliant
blue R-250; (B) alginate lyase activity detected on a gel containing
1% alginic acid. Lanes: M, molecular mass markers; 1, periplasmic
fraction (10 µg); 2, hydroxyapatite column AlgL fraction (10 µg);
3, phenyl-Sepharose AlgL fraction (10 µg).
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In agreement with other reports, which have described the partial
purification of AlgL from both
A. chroococcum and
A. vinelandii (
23), attempts to purify the native protein
to homogeneity were
unsuccessful even though a variety of purification
steps were
used. In this case, the recovery of partially purified
enzyme
(0.06 mg/liter of culture) was also lower than the obtained with
the overexpressed alginate lyase (0.36 mg/liter).
To characterize the purified enzyme, it was found that the optimum
temperature for the recombinant enzyme assay is 30°C at
pH 7.5 and
that alginate lyase was inactive when analyzed at 70°C
(Fig.
5). These values are rather similar to
those for the native
enzyme (data not shown).
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FIG. 5.
Optimum temperature for the recombinant alginate lyase
assay. Aliquots (25 µl) of an enzyme preparation (1.5 mU/µl) were
analyzed for activity at the indicated temperatures, and the values
found after a 30-min assay were spectrographically recorded at 548 nm
by the thiobarbituric acid method.
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The enzyme activity was measured in the presence of cations at 100 mM.
Like the native protein (
35), the activity of recombinant
AlgL increased in the presence of Na
+ and K
+;
the combined action of these cations and Mg
2+ increased the
activity 100-fold with respect to the control without
added cations
(Table
2). When these monovalent and
divalent cations
were used at concentrations in the range from 1 to 50 mM, the
increase in alginate lyase activity was not significant. Other
cations, like Ca
2+, Co
2+, Mn
2+, and
Zn
2+ or the presence of EDTA does not affect the enzymatic
activity.
This enhancing effect of Mg
2+ on alginate lyase
activity has been reported for the enzyme from
Klebsiella
aerogenes (
25),
P. alginovora
(
7), and a marine
bacterium that has not been identified
(
36). In all these cases,
the presence of Mg
2+
is stimulative but is not essential for the activity. There are
other
situations, however, where the cations are strictly required.
Thus,
Bacillus circulans JBH2 alginate lyase requires
Mg
2+ (
18), and a
Pseudomonas sp.,
P. aeruginosa, and
Littorina sp.
alginate lyase
depends on Ca
2+ (
16). The enzyme from
A. chroococcum 4A1M is activated by Ca
2+ and inhibited
strongly by Hg
2+ (
19). It should be mentioned
that the enzyme from
A. chroococcum 4A1M has a molecular
mass of 23 kDa by SDS-PAGE and 24 kDa by
gel filtration. Therefore, its
molecular mass is roughly half
that of the enzyme found in, and
isolated by us from,
A. chroococcum ATCC 4412. Furthermore,
the enzyme from strain 4A1M showed maximum
activity at 60°C whereas
the one used in the present work has
maximum activity at 30°C.
The
Km value for the recombinant
A. chroococcum alginate lyase was found to be 0.08 mM for uronic
acids, which is in the range
from 0.1 to 0.5 mM reported for other
alginate lyases (
16).
From a Lineweaver-Burk
double-reciprocal plot, a
Vmax value of
0.183 mM
min
1 was calculated for the
enzyme.
To study the substrate specificity, the recombinant alginate lyase was
used to degrade a series of alginates and the activity
was assayed.
AlgL was 17-fold more active against alginate from
the seaweed
M. pyriferia than against that from
P. aeruginosa.
The
enzymatic activity against the
A. chroococcum alginate was
lower than with both above-mentioned substrates. Alginate from
P. aeruginosa was 37% acetylated, but that from
M. pyriferia was
completely deacetylated. Therefore, it is likely
that the acetylation
of the bacterial alginate confers some protection
against depolymerization
by alginase. In favor of this conclusion is
the fact that after
chemical deacetylation of the
Pseudomonas alginate,
A. chroococcum AlgL
increased its activity around 10-fold.
Finally, the possible roles of alginate lyases in some environments and
several potential applications for both the oligosaccharides
derived
from the action of alginase and the enzyme itself have
recently been
discussed (
41). Along these lines, Murata et al.
(
32) reported that they obtained an alginate lyase in large
quantity and suggested its possible value as a therapeutic agent
for
the treatment of cystic fibrosis patients infected with mucoid
P. aeruginosa. In fact, the use of alginate lyases to degrade
the
alginate in the lungs of cystic fibrosis patients, allowing
a major
diffusion of antibiotic, has been recently reported (
20).
The alginate lyase from
A. chroococcum could be used as
therapeutic
agent because, as mentioned above, it is able to degrade
the alginate
produced by
P. aeruginosa.
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ACKNOWLEDGMENTS |
This work was supported by financial help from Plan Andaluz de
Investigación and Consejo Superior de Investigaciones
Científicas (Spain). A. Pascual was supported by a fellowship
from the Spanish Ministry of Education.
We thank F. J. Cejudo and J. de la Cruz for critical reading of
the manuscript.
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FOOTNOTES |
*
Corresponding author. Mailing address: Instituto de
Bioquímica Vegetal y Fotosíntesis, Centro de
Investigaciones Científicas "Isla de la Cartuja," Avda.
Américo Vespucio s/n, E-41092 Seville, Spain. Phone: 34 95 448 95 25. Fax: 34 95 446 00 65. E-mail: apguer{at}cica.es.
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REFERENCES |
| 1.
|
Ausubel, F.,
R. Brent,
R. Kingston,
D. Moore,
J. Seidman, and K. Struhl.
1989.
Current protocols in molecular biology.
Greene Publishing and Wiley-Interscience, New York, N.Y.
|
| 2.
|
Becking, J. H.
1981.
The family Azotobacteraceae, p. 794-817.
In
M. P. Starr, H. Stolp, H. G. Trüper, A. Balows, and H. G. Schlegel (ed.), The prokaryotes. Springer-Verlag KG, Berlin, Germany.
|
| 3.
|
Boyd, A., and A. M. Chakrabarty.
1994.
Role of alginate lyase in cell detachment of Pseudomonas aeruginosa.
Appl. Environ. Microbiol.
60:2355-2359[Abstract/Free Full Text].
|
| 4.
|
Boyd, A.,
M. Ghosh,
T. B. May,
D. Shinaberger,
R. Koegh, and A. M. Chakrabarty.
1993.
Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product.
Gene
131:1-8[Medline].
|
| 5.
|
Boyd, J., and J. R. Turvey.
1977.
Isolation of a poly--L-guluronate lyase from Klebsiella aerogenes.
Carbohydr. Res.
57:163-171[Medline].
|
| 6.
|
Boyd, J., and J. R. Turvey.
1978.
Structural studies of alginic acid using a bacterial poly--L-guluronate lyase.
Carbohydr. Res.
66:187-194.
|
| 7.
|
Boyen, C.,
Y. Bertheau,
T. Barbeyront, and G. B. Kloare.
1990.
Preparation of guluronate lyase from Pseudomonas alginovora for protoplast isolation in Laminaria.
Enzyme Microb. Technol.
12:885-890.
|
| 8.
|
Bradford, M.
1976.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:248-254[Medline].
|
| 9.
|
Cote, G. L., and L. H. Krull.
1988.
Characterization of the exocellular polysaccharides from Azotobacter chroococcum.
Carbohydr. Res.
181:143-152.
|
| 10.
|
Devereux, J.,
P. Haeberli, and O. Smithies.
1984.
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res.
12:387-395.
|
| 11.
|
Efterkhar, F., and N. L. Schiller.
1994.
Partial purification and characterization of a manuronan-specific alginate lyase from Pseudomonas aeruginosa.
Curr. Microbiol.
29:37-42.
|
| 12.
|
Elhai, J., and C. Wolk.
1988.
A versatile class of positive-selection vectors based on the nonviability of palindrome containing plasmid that allows cloning into long polylinker.
Gene
68:119-138[Medline].
|
| 13.
|
Fialho, A. M.,
N. A. Zielinski,
W. F. Fett,
A. M. Chakrabarty, and A. Berry.
1990.
Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotobacter lineage of superfamily B procaryotes.
Appl. Environ. Microbiol.
56:436-443[Abstract/Free Full Text].
|
| 14.
|
Franklin, M. J., and D. E. Ohman.
1996.
Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation.
J. Bacteriol.
178:2186-2195[Abstract/Free Full Text].
|
| 15.
|
Gacesa, P.
1988.
Alginates.
Carbohydr. Polym.
8:161-182.
|
| 16.
|
Gacesa, P.
1992.
Enzymic degration of alginates.
Int. J. Biochem.
24:545-552[Medline].
|
| 17.
|
Gacesa, P., and F. S. Wusteman.
1990.
Plate assay for simultaneous detection of alginate lyase and determination of substrate specificity.
Appl. Environ. Microbiol.
56:2265-2267[Abstract/Free Full Text].
|
| 18.
|
Hansen, J. B.,
R. S. Doubet, and J. Ram.
1984.
Alginase enzyme production by Bacillus circulans.
Appl. Environ. Microbiol.
47:704-709[Abstract/Free Full Text].
|
| 19.
|
Haraguchi, K., and T. Kodama.
1996.
Purification and properties of poly(-D-mannuronate) lyase from Azotobacter chroococcum.
Appl. Microbiol. Biotechnol.
44:576-581.
|
| 20.
|
Hatch, R. A., and N. L. Schiller.
1998.
Alginate lyase promotes diffusion of aminoglycosides through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa.
Antimicrob. Agents Chemother.
42:974-977[Abstract/Free Full Text].
|
| 21.
|
Haug, A.,
B. Larsen, and E. Baardseth.
1969.
Comparison of the constitution of alginates from differents sources.
Proc. Int. Seaweed Symp.
6:443-451.
|
| 22.
|
Jarman, T. R.,
L. Deavin,
S. Scolombe, and R. C. Righelato.
1978.
Investigation of the effect of environmental conditions on the rate of exopolysaccharide synthesis in Azotobacter vinelandii.
J. Gen. Microbiol.
107:59-64.
|
| 23.
|
Kennedy, L.,
K. McDowell, and I. W. Sutherland.
1992.
Alginases from Azotobacter species.
J. Gen. Microbiol.
138:2465-2471.
|
| 24.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[Medline].
|
| 25.
|
Lange, B.,
J. Wingender, and U. K. Winkler.
1989.
Isolation and characterization of an alginate lyase from Klebsiella aerogenes.
Arch. Microbiol.
152:302-308[Medline].
|
| 26.
|
Lawson, G. L., and M. Stacey.
1954.
Immunopolysaccharides. I. Preliminary studies of a polysaccharide from Azotobacter chroococcum containing an uronic acid.
J. Chem. Soc.
1954:1925-1931.
|
| 27.
|
Maki, H.,
A. Mori,
K. Fujiyama,
S. Kinoshita, and T. Yoshida.
1993.
Cloning, sequence analysis and expression in Escherichia coli of a gene encoding an alginate lyase form Pseudomonas sp. OS-ALG-9.
J. Gen. Microbiol.
139:987-993[Abstract/Free Full Text].
|
| 28.
|
Malissard, M.,
C. Duez,
M. Guinand,
M.-J. Vacheron,
G. Michel,
N. Marty,
B. Joris,
T. Thamm, and J.-M. Ghuysen.
1993.
Sequence of a gene encoding a (polyManA) alginate lyase active on Pseudomonas aeruginosa alginate.
FEMS Microbiol. Lett.
110:101-106[Medline].
|
| 29.
|
May, T. B., and A. M. Chakrabarty.
1994.
Isolation and assay of Pseudomonas aeruginosa alginate.
Methods Enzymol.
235:295-304[Medline].
|
| 30.
|
Meissner, P. S.,
W. P. Sisk, and M. L. Berman.
1987.
Bacteriophage 1 cloning system for the construction of directional cDNA libraries.
Proc. Natl. Acad. Sci. USA
84:4171-4175[Abstract/Free Full Text].
|
| 31.
|
Monday, S. R., and N. L. Schiller.
1996.
Alginate synthesis in Pseudomonas aeruginosa: the role of AlgL (alginate lyase) and AlgX.
J. Bacteriol.
178:625-632[Abstract/Free Full Text].
|
| 32.
|
Murata, K.,
T. Inose,
T. Hisano,
S. Abe,
Y. Yonemoto,
T. Yamashita,
M. Takagi,
K. Sakaguchi,
A. Kimura, and T. Imanaka.
1993.
Bacterial alginate lyase: enzymology, genetics and application.
J. Ferment. Bioeng.
76:427-437.
|
| 33.
|
Pearson, W., and D. Lipman.
1988.
Improved tools for biological sequence comparison.
Proc. Natl. Acad. Sci. USA
85:2444-2448[Abstract/Free Full Text].
|
| 34.
|
Peciña, A., and A. Paneque.
1994.
Detection of alginate lyase by activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent renaturation.
Anal. Biochem.
217:124-127[Medline].
|
| 35.
|
Peciña, A.
1997.
Ph.D. thesis.
University of Seville, Seville, Spain.
|
| 36.
|
Romeo, A., and J. F. Preston.
1986.
Purification and structural properties of an extracellular (1 4)--D-mannuronan specific alginate lyase from a marine bacterium.
Biochemistry
25:8385-8391.
|
| 37.
|
Sambrook, J.,
E. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 38.
|
Sanger, F.,
S. Nicklen, and A. Coulson.
1977.
DNA sequencing with chain-terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 39.
|
Schiller, N. L.,
S. R. Monday,
C. M. Boyd,
N. T. Keen, and D. E. Ohman.
1993.
Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli.
J. Bacteriol.
175:4780-4789[Abstract/Free Full Text].
|
| 40.
|
Studier, F. W., and B. A. Moffatt.
1986.
Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of clones genes.
J. Mol. Biol.
189:113-130[Medline].
|
| 41.
|
Sutherland, I. W.
1995.
Polysaccharide lyases.
FEMS Microbiol. Lett.
16:323-347.
|
| 42.
|
Weissbach, A., and J. Hurwitz.
1958.
The formation of 2-keto-3-deoxy heptonic acid in extracts of Escherichia coli B. I and II.
J. Biol. Chem.
234:705-712.
|
Journal of Bacteriology, March 1999, p. 1409-1414, Vol. 181, No. 5
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
