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Applied and Environmental Microbiology, November 2001, p. 5197-5203, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5197-5203.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Purification and Properties of a Glucuronan Lyase
from Sinorhizobium meliloti M5N1CS (NCIMB
40472)
Alexandre
Da Costa,1
Philippe
Michaud,1
Emmanuel
Petit,1
Alain
Heyraud,2
Philippe
Colin-Morel,2
Bernard
Courtois,1 and
Josiane
Courtois1,*
Laboratoire des Polysaccharides Microbiens et
Végétaux, IUT, Département de Génie Biologique,
Université de Picardie Jules Verne, Avenue des Facultés, Le
Bailly, 80025 Amiens Cedex,1 and
CERMAV-CNRS Université Joseph Fourrier, 38041 Grenoble
Cedex,2 France
Received 10 April 2001/Accepted 4 September 2001
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ABSTRACT |
A glucuronan lyase extracted from Sinorhizobium
meliloti strain M5N1CS was purified to homogeneity by
anion-exchange chromatography. The purified enzyme corresponds to a
monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific
activity was found only for polyglucuronates leading to the production
of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal
at pH 6.5 and 50°C. Zn2+, Cu2+, and
Hg2+ (1 mM) inhibited the enzyme activity. No homology of
the enzyme N-terminal amino acid sequence was found with any of the
previously published protein sequences. This enzyme purified from
S. meliloti strain M5N1CS corresponding to a new lyase was
classified as an endopolyglucuronate lyase.
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INTRODUCTION |
Bacterial polysaccharides as well as
plant polysaccharides can be degraded by specific glycosidases
corresponding either to hydrolases or to lyases. Among the polyanionic
polymers, some of them (i.e., alginate) are exclusively degraded by
lyases (35, 40, 42). The lyase cleaving mechanism consists
of a 
-elimination, in which a general base-catalyzed abstraction of
the proton at C-5 of a uronic acid occurs. A transfer of electrons from
the carboxyl group to form a double bond between C-4 and C-5 results in
the elimination of the 4-O-glycosidic bond and in the
formation of
4-deoxy-L-erythro-hex-4-enopyranosyluronic acid.
This reaction leads to the formation of an unsaturated uronate at the
newly generated, nonreducing end. Oligosaccharides from a degree of polymerization of 2 to 3 or 5 can be obtained by polysaccharide degradation with lyases (3, 20, 40). To date, lyase
activities on various polymers have been described as alginate
(16, 32, 35), gellan (41), hyaluronan
(39), ulvan (25), and xanthan (15). The tested lyases were found in many organisms such
as marine gastropods (19) or fungi (36, 45)
as well as in many bacteria (14, 15, 31) and
bacteriophages (1, 4). The enzyme localization in the
producing organisms may be in the cytosol (28, 36) and the
periplasmic space (22), as for alginate lyase from
Azotobacter species, or in extracellular fractions, as for a
Bacillus circulans lyase (26). Lyases present
possible applications in the medical field, such as, for example, the
alginate lyase, which promotes diffusion of antibiotics through
extracellular polymers produced by the pathogenic
Pseudomonas sp. (17). Due to potential
applications, studies concerning polysaccharide lyases have been increasing.
The Sinorhizobium meliloti mutant strain M5N1CS (NCIMB
40472) (7), which induces the formation of effective
nodules on alfalfa roots (12), produces a glucuronan
(6, 18) corresponding to high-molecular-weight (HMW) and
low-molecular-weight (LMW) (1
4)--D-polyglucuronic
acid partially acetylated at the C-2 and/or C-3 position. The detection
of LMW glucuronan containing a 4-5 unsaturated glucuronic residue at
the nonreducing end (29, 30) led us to suspect a polymer
degradation by a glucuronan lyase.
In order to produce oligoglucuronans, a lyase degrading various
acetylated and nonacetylated glucuronans was purified and characterized.
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MATERIALS AND METHODS |
Bacterial strain and culture conditions.
For glucuronan
lyase production, S. meliloti strain M5N1CS (NCIMB 40472)
(6) was aerobically cultured in a 2-liter Erlenmeyer flask
containing 1 liter of tryptone yeast (TY) medium (pH 7.2) (2) supplemented with sucrose (1% [wt/vol]) (TYS),
during 75 h at 30°C on a rotary shaker (100 rpm). The inoculum
was 100 ml of a S. meliloti M5N1CS suspension in the same
TYS medium, grown 20 h at 30°C under agitation (100 rpm). A
10-ml bacterial suspension grown 20 h at 30°C was used to
inoculate the 100-ml TYS medium.
Glucuronan production.
S. meliloti strain M5N1CS
was cultivated at 30°C in a 6-liter reactor (LSL Biolaffite,
Saint-Germain-en-Laye, France) containing 4.5 liters of
Rhizobium Complete medium (5) supplemented with sucrose (1% [wt/vol]) (RCS). The inoculum was 0.5 liter of RCS in a
1-liter Erlenmeyer flask inoculated with S. meliloti M5N1CS. After 20 h of incubation on a rotative shaker (100 rpm) at 30°C, the inoculum was transferred to the reactor and incubated as previously described (29), with or without addition of 0.15%
(wt/vol) MgSO4 · 7H2O for the production
of mainly 3-O-acetylated (standard) or
2,3-di-O-acetylated (highly acetylated) glucuronans.
Glucuronan isolation, purification, and characterization.
Two HMW glucuronans, one which was standard (mainly
3-O-acetylated) and one which was highly acetylated (mainly
2,3-di-O-acetylated), were produced during two 75-h
fermentations. Then the broths were centrifuged (33,900 × g, 40 min, 20°C), and the HMW polysaccharides were concentrated
from the cell-free broth by tangential ultrafiltration on a
105 normal-molecular-weight cutoff (NMWCO) polysulfone
membrane (0.1 m2) (Sartorius, Göttingen, Germany).
The concentrates were diluted with 1 volume of distilled water and
ultrafiltered as above; this step was repeated five times, and then the
last concentrated products were dried under vacuum. The substitution
degree determined by 1H nuclear magnetic resonance (NMR)
spectroscopy was one acetate for 1.3 residue and one acetate for 0.7 residue for the standard and highly acetylated glucuronans,
respectively (8). A fraction of the standard glucuronan
was completely deacetylated by 2 M KOH during 12 h at 50°C (pH
12). The complete deacetylation of glucuronan was confirmed by
1H NMR spectroscopy.
Preparation of a crude enzyme fraction.
All steps were
carried out at 4°C. At the end of the incubation (75 h) the bacterial
suspension was centrifuged (13,880 × g, 30 min), and
the pellet was collected and suspended in 10 mM Tris-HCl buffer (pH
8.0) (final volume, 80 ml). The bacterial suspension was disrupted with
an ultrasonicator (Vibra-Cell model VCX 600 W; Danbury, Conn.) at 20 kHz (13-mm-diameter standard probe) for 30 min, and then the mixture
was centrifuged at 50,000 × g for 1 h. The
supernatant, called crude enzyme fraction, was collected and stored at
80°C.
Enzyme purification.
The enzyme was purified at room
temperature, through a two-step procedure using low-pressure liquid
chromatography (ProTeam LC System 210; Isco, Lincoln, Nebr.).
(i) Step 1.
The purification was performed on a ready-to-use
anion exchanger Sartobind membrane adsorber (MA) Q100 (Sartorius,
Goettingen, Germany), which had first been equilibrated with 10 mM
Tris-HCl buffer (pH 8.0). Ten milliliters of crude enzyme fraction was loaded onto the Sartobind cartridge. The Sartobind MA unit was first
washed with the equilibration buffer (112 ml) with a flow rate of 4.3 ml/min; then the eluent consisted of a KCl gradient from 0 to 50 mM in
30 s and a 50 mM KCl buffer (69 ml) at the same flow rate as
previously. A KCl buffer (at least 100 mM) was used. Fractions (8.6 ml)
were collected with a Foxy 200 collector (Isco). Enzymatic activity was
tested on each fraction as described below. Fractions presenting a
glucuronan lyase activity were pooled and ultrafiltered in a stirred
Amicon cell (Beverly, Mass.) through a 104 NMWCO
polyethersulfone membrane (Sartorius). The retentate dispersed in the
buffer used for step 2 (10 mM Tris-HCl [pH 7.2]) was ultrafiltered as
previously (this step was repeated twice) and concentrated to a final
volume of 1.3 ml.
(ii) Step 2.
The concentrated enzyme solution from step 1 was applied as described above to the Sartobind MA, which had first
been equilibrated with a 10 mM Tris-HCl buffer (pH 7.2). The Sartobind
MA unit was then washed with the equilibration buffer (129 ml), and a
KCl gradient (ranging from 0 to 30 mM in 30 s) was applied.
Proteins were eluted with a 30 mM KCl buffer (77 ml). The flow rate was as above. Fractions presenting a glucuronan lyase activity were pooled,
washed, concentrated by ultrafiltration to a final volume of 2 ml, and
stored at 80°C.
Enzyme assays.
During the glucuronan lyase purification
steps, the enzyme was tested on the reaction mixture consisting of 1 ml
of 10 mM Tris-HCl buffer (pH 7.2), 0.5 ml of glucuronan in 10 mM
Tris-HCl buffer (pH 7.2) (0.2% [wt/vol]) and 0.5 ml of enzyme
solution. A fraction aliquot, which was immediately frozen, (80°C)
corresponded to the standard (t = 0 h). The
remaining reaction mixture was incubated 15 h at 30°C on an HS
500 shaker (Jankel & Kunkel, Staufen, Germany). The
polysaccharide degradation was determined by quantitative analysis of
the reducing sugar, using the 2,2'-bicinchoninate method
(43). The lyase activity was confirmed by measuring
A235 arising from the double bond in the
reaction product, on a Uvikon 930 spectrophotometer (Kontron, Montigny
Lebretonneux, France). One unit (U) of the enzyme activity was defined
as an increase of 1 unit per h in the absorbance at 235 nm. Specific
activity was expressed as units per milligram of protein.
The glucuronan lyase relative activity was determined on 60 µl of the
deacetylated glucuronan (1.3 g/liter) in 50 mM
KH2PO4 buffer (pH 6.5) mixed to 20 µl of the
purified glucuronan lyase. Then, the mixture was incubated at room
temperature for 2 h. Analytical chromatographic separation of the
oligo-uronides obtained by enzymatic hydrolysis of deacetylated
glucuronan was performed on a Millipore Waters Model 510 high-performance liquid chromatography apparatus, using two
high-performance gel filtration columns in sequence: an OHpak
B-805 (8 by 500 mm) from Shodex (Tokyo, Japan) and a TSKgel
G3000PW 10 µm (7.5 by 300 mm) from Interchim (Montluçon, France). Chromatography was run isocratically at room temperature, with
0.1 M NaNO3 as eluent (1 ml/min). Detection was performed online with a R-410 Waters differential refractometric detector. Chromatograms were analyzed and recorded with a C-R4A chromatopac integrator (Shimadzu). Glucuronan lyase relative activity was estimated
by comparison of chromatograms obtained with the substrate alone and
after enzymic hydrolysis.
Protein determination.
Protein in Sartobind MA fractions was
routinely monitored by measuring A280 with a
UA-6 UV detector (Isco). The amounts of protein in the crude and
purified enzyme fractions were estimated with a bicinchoninic acid
protein assay; bovine serum albumin was used as a standard
(38).
SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed in a vertical slab unit (Mini
Protean II Slab Cell; Bio-Rad, Richmond, Calif.) according to the
procedure described by Laemmli (23). The separating gel
(18%) was overlaid with a stacking gel (4%). The samples were
incubated in 50 mM Tris-HCl (pH 7.5), with 1% (wt/vol) SDS and 1%
(vol/vol) 2-mercaptoethanol. Protein samples (from 28.5 to 107 µg)
and a standard protein mixture (from 14,400 to 97,400 Da) (Bio-Rad)
were applied to the gel. The electrophoresis was performed for 15 h at room temperature at 80 V. Gels were stained with Coomassie
brilliant blue R-250 (Bio-Rad).
IEF.
Analytical isoelectric focusing (IEF) was performed on
a 5% IEF polyacrylamide gel plate (Bio-Rad) with a broad-range
ampholyte (pI ranging from 3 to 10). A protein calibration mix with pI
values from 4.45 to 9.6 was used. The IEF was performed for 1 h at room temperature at 100 V, and then 250 V was applied during 1 h, and at least 500 V was applied during 30 min. Proteins were stained with an
IEF gel staining solution (Bio-Rad).
Determination of the N-terminal sequence.
N-terminal amino
acid sequence of the purified lyase was determined by Edman degradation
(10) with a Procise 494 protein sequencing system (Applied
Biosystems, Division of Perkin-Elmer).
Fractionation of oligoglucuronates.
Oligoglucuronates
obtained by enzymatic degradation were size fractionated by preparative
gel filtration on a glass chromatographic column (2.5 by 100 cm) packed
with Bio-Gel P-6 (Bio-Rad), eluted with a 50 mM NaNO3
solution, with a flow rate of 50 ml/h. Detection was achieved with a
R-403 Waters differential refractometer. Fractions (10 ml) were
collected with a 201-202 model Gilson collector. Fractions belonging
to a same peak were combined, concentrated, desalted by
high-performance liquid chromatography on a Toyopearl HW 40F/50F
(Interchrom) column, and freeze-dried.
NMR.
1H NMR analyses were performed at 85°C
with an AC-300 Bruker (Bruker Spectrospin, Wissembourg, France) Fourier
transform spectrometer with a 5-mm 1H 13C dual
probe. To equilibrate exchangeable protons, the different samples (20 mg) were freeze-dried and then dispersed in 500 µl of
D2O. 1H NMR spectra were obtained using a
spectral width of 300 MHz, a 16K data-block size, and a pulse sequence
of 8 µs; 16 scans with acquisition of 2.73 s were
accumulated. The H2O signal was presaturated using the
standard Bruker Presat sequence, with a delay of 3 s and a
decoupler power of 20 dB at low range. The polymerization degree was
estimated by comparison of the integral of the H-1 or H-4 signals of
the unsaturated unit to the integral of the other H-1 signals
(9).
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RESULTS |
Glucuronan lyase purification.
Four liters of a 75-h S. meliloti M5N1CS culture broth was centrifuged (13,880 × g, 30 min). The pellet was suspended in 80 ml of a 10 mM
Tris-HCl buffer (pH 8.0) and sonicated. After centrifugation (50,000 × g, 1 h) the supernatant corresponding
to the crude enzyme fraction was collected and tested on native and
deacetylated glucuronans. Polymer degradation was monitored using the
reducing sugar assay (43) (Fig.
1). We noted that the deacetylated
glucuronan was the better substrate for lyase activity. As the
thiobarbituric acid method (44) routinely used for lyase
activity studies leads to the formation of glucuronan precipitates, in
this study, the specific lyase activity was determined by UV
spectrophotometry (
= 235 nm). We noted that the specific
activity on the crude enzyme fraction was 4 × 10
2
U/mg.
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FIG. 1.
Evolution of reducing sugar concentration during
incubation of S. meliloti M5N1CS cell extracts with
deacetylated (  ) and native ( ) glucuronan fractions
(expressed in millimolar glucuronic acid equivalents). Each data point
represents the mean of at least three values, which did not vary more
than 15% from the mean.
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The enzyme purification was performed first with a Sartobind MA Q100
unit by injection of the whole crude enzyme fraction
(176 ml) through
injections of 10 ml. In the first purification
step, the Sartobind MA
was washed with 112 ml of the 10 mM Tris-HCl
buffer (pH 8.0). Then, the
elution was performed with KCl buffers
(50 and 100 mM). Three fractions
absorbing at 280 nm were identified
from the crude enzyme fraction
(Fig.
2A): the first one eluted
with the
equilibration buffer, the second one eluted with the
50 mM KCl buffer,
and the third eluted with the 100 mM KCl buffer.
The glucuronan lyase
activity was detected only in the fraction
eluted with 50 mM KCl. The
fraction from 120.4 to 137.6 ml was
collected, desalted, and
concentrated. The activity of the glucuronan
lyase chromatographied
fraction (noted GLC1) was increased 23-fold.
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FIG. 2.
Elution profiles on a Sartobind Q100 MA of the crude
enzyme fraction from S. meliloti M5N1CS (step 1) (A) and
from GLC1 fraction (step 2) (B). Membranes were equilibrated with 10 mM
Tris-HCl buffer at pH 8 (A) and pH 7.2 (B). Elution was carried out
first with the equilibration buffer, then with 50 mM KCl buffer for
step 1 (A), and then with 30 mM KCl buffer for step 2 (B). Glucuronan
lyase (GL) activity ( ), KCl concentration
(---), and absorbance at 280 nm ().
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In order to achieve the glucuronan lyase purification, the GLC1
fraction was applied on the same Sartobind MA unit as above.
In the
second purification step, the membrane was washed with
129 ml of 10 mM
Tris-HCl buffer (pH 7.2) and eluted with 30 and
100 mM KCl buffers
(Fig.
2B). The active fraction (eluted with
the 30 mM KCl buffer from
146.2 ml to 163.4 ml) was desalted,
concentrated by ultrafiltration to
a final volume of 2 ml, and
stored at
80°C. At this level of
purification, the activity of
the glucuronan lyase fraction (noted
GLC2) was purified 394-fold
compared to the crude enzyme fraction, with
a yield of 2.5%. We
noted that the glucuronan lyase activity remained
constant in
samples stored at
80°C for several weeks (data not
shown).
Basic features of purified glucuronan lyase.
The purified
enzyme preparation was examined by SDS-PAGE in denaturing conditions
(Fig. 3). The GLC2 fraction was
identified as a single protein band (lane D), while the GLC1 fraction
(lane C) revealed numerous protein bands. The purification procedures applied to the bacterial extracts revealed to be selective for the
purification of a 20-kDa protein exhibiting a glucuronan lyase activity. The GLC2 fraction analyzed by IEF revealed a single band with
pI estimated at 4.9 (Fig. 4, lane A).
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FIG. 3.
SDS-PAGE of glucuronan lyase fractions at different
purification steps, after staining with Coomassie brilliant blue R-250.
Lanes: A, molecular mass marker proteins; B, crude enzyme fraction; C,
GLC1 fraction; D, GLC2 fraction.
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FIG. 4.
Analytical IEF of purified glucuronan lyase from
S. meliloti M5N1CS. Lanes: A, purified glucuronan lyase; B,
pI marker proteins. The proteins were stained with the IEF gel staining
solution.
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Effects of environmental conditions.
The enzyme activity was
investigated under different buffer conditions at room temperature. The
lyase activity tested in KH2PO4 buffers (pH
6.5), ranging from 10 to 200 mM (data not shown), was quite similar to
those obtained in glycine-sodium hydroxyde and potassium phosphate
buffers at pH ranging from 7 to 8. The 50 mM
KH2PO4 buffer at pH 6.5 was used in all further
experiments. The glucuronan lyase activity tested between 6 and 79°C
in a 50 mM KH2PO4 buffer revealed a maximal
relative activity at 50°C. This activity was reduced by one-half at
52°C. Lyase activity tested on heated enzyme fractions in the 50 mM
KH2PO4 buffer indicated that the enzyme was
stable at up to 50°C and decreased at higher temperatures. The enzyme
was completely inactivated at 67°C. The lyase activity was tested on
enzyme samples (in 50 mM KH2PO4 buffer at pH
6.5) incubated previously with 1 mM metal salts for 2 h at 50°C.
Under the conditions tested, no effect was observed with magnesium
(MgSO4), whereas sodium (NaN3), cadmium
[Cd(NO3)2], and lithium (LiCl) reduced
activity by 26 to 29%, and similar reductions were observed with
manganese (MnSO4), EDTA, and calcium (CaCl2)
(21 to 30%). However, the enzyme proved to be very sensitive to zinc
(ZnCl2) (59% reduction), copper (CuCl2)
(85%), mercury (HgCl2) (94%), and silver
(AgNO3) (100% reduction).
N-terminal amino acid sequence.
From the purified enzyme, the
sequence of 17 amino acids at the N-terminal site was identified as
A-E-I-K-D-P-E-N-T-I-L-M-E-T-T-K-G. A comparison of the
N-terminal amino acid sequence from the S. meliloti M5N1CS
lyase to protein sequences of the GenBank coding sequence data
(398,151 sequences searched) revealed no homology with any previously
published protein sequences.
Substrate specificity of glucuronan lyase.
Three glucuronan
substrates (0.4% [wt/vol] in the incubation buffer), corresponding
to a deacetylated glucuronan, a mainly 3-O-acetylated
glucuronan, and a mainly 2,3-di-O-acetylated glucuronan, were incubated at 50°C overnight with the enzyme preparation. The
polymer degradation was monitored by size exclusion chromatography of
the reaction mixture. As described in Table
1, the enzyme was highly specific to
deacetylated glucuronan; a smaller activity was noted with the mainly
3-O-acetylated glucuronan (standard), while the mainly
2,3-di-O-acetylated glucuronan (highly acetylated) was not
cleaved.
Degradation products from deacetylated glucuronan.
A
deacetylated glucuronan solution (2% [wt/vol]) was incubated with
0.46 U of enzyme. After 15 h of incubation the solution was
chromatographed on a Bio-Gel P-6 column, and the elution profile was
compared to those obtained with a sample corresponding to a
deacetylated glucuronan solution as above, incubated without enzyme. No
oligosaccharides were detected with the enzyme-free sample, while
oligoglucuronates with degrees of polymerization (dp) ranging from 2 to
>7 were separated (Fig. 5).
1H NMR spectra from each oligoglucuronate fractions
obtained after enzymatic degradation of the glucuronan sample (Fig.
6) revealed doublets at 5.68 ppm,
characteristic of H-4 of an unsaturated unit, and signals at 4.96 and
4.32 ppm assigned to H-1 of an unsaturated residue and to H-1 of the
repeating unit. The pure oligoglucuronans obtained (dp, 2 to 7),
containing an unsaturated residue at the non-reducing end, were
significative of a randomly cleaving enzyme.
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FIG. 5.
Gel permeation analysis of deacetylated glucuronan
incubated with S. meliloti M5N1CS glucuronan lyase, under
the following conditions: by Bio-Gel P-6 column, 100 by 2.5 cm; flow
rate, 50 ml/h; eluent, 50 mM NaNO3; refractive index
detection. The degrees of polymerization are indicated by numbers under
the peaks.
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FIG. 6.
1H NMR spectrum of deacetylated
oligoglucuronan (dp 5) in D2O (300 MHz; 50°C). H4//, H4
of the unsaturated non-reducing terminus; H1, H1 of the reducing
end unit; H1 , H1 of the reducing end unit; H1, H1 of the
non-terminus units; H1//, H1 of the unsaturated non-reducing
terminus.
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DISCUSSION |
S. meliloti mutant strain M5N1CS (NCIMB 40472) produces
during growth a homopolymer of (14)--D-glucuronic
acid partially acetylated. This exopolysaccharide may have a
protecting role, as does EPS II, which is produced by Rhizobium
meliloti EFBI (24, 27). In previous works
(29), it was noted that the weight-average molecular weight (Mw) of the glucuronan
decreased from 7.5 × 105 to 5 × 105
between 27 and 75 h of fermentation. The polymer
Mw reduction was correlated with the formation
of LMW (from 103 to 5 × 104) glucuronans,
whose 1H NMR spectra revealed a doublet at 5.85 ppm
characteristic of the H-4 of an unsaturated unit corresponding to the
non-reducing terminus unit of the glucuronan. This result was
associated to a lyase activity first detected in cell extracts
(30). After purification of the enzyme, we determined that
the N-terminal amino acid sequence revealed no homology to published
protein sequences; the lyase extracted from S. meliloti
strain M5N1CS corresponds to a new enzyme. We noted a relative
homogeneity between the pI values of specific alginate lyases (from 4.7 to 5.1) (11, 35) and those of the glucuronan lyase (4.9).
We noted that addition of magnesium (MgSO4 · 7H2O; 1 mM) to enzyme samples has no effect on the
glucuronan degradation, while the addition of magnesium (from 10 to
16 mM MgSO4 · 7H2O) to bacterial
suspensions during fermentations leads to glucuronan degradations
(29). The polymer degradation may be due to the release of
periplasmic proteins as the glucuronan lyase, from bacteria, during
magnesium sulfate additions. The glucuronan lyase was tested on uronic
acid containing polymers from plants (polygalacturonate and
polygalacturonate methyl ester, guluronate, and mannuronate), from
animals (hyaluronate), or from bacteria (glucuronan and glucoglucuronan
[13]); we noted that under the conditions applied the
enzyme cleaved only deacetylated and mainly 3-O-acetylated
glucuronans. However, the yield of acetyl residues present on the
polymer influences the lyase activity. No degradation was detected when
the substrate was a highly acetylated glucuronan (one acetyl for 0.7 residue), while lowly acetylated (mainly 3-O-acetylated) and
deacetylated glucuronans were degraded. These results confirm that
oligoglucuronans with dp 
2, containing no
2,3-di-O-acetylated residues, detected in fermentation media of S. meliloti mutant strain M5N1CS correspond to
degradation products of unacetylated and monoacetylated glucuronan
sequences (33) present on the polymer (8).
These results agree with those presented by Skjak-Braek et al.
(37), who proposed that acetylation of mannurosyl residues
on positions C-2 and C-3 inhibited the alginate lyase activity from
different Azotobacter species. Similar results were
presented by Kennedy et al. (22) with alginate lyases
tested on O-acetylated mannuronate blocks. Pure
oligoglucuronan fractions with dp of 2 to 7 were obtained by
degradation of a deacetylated glucuronan with the purified enzyme, and
unsaturated residues were detected on 1H NMR spectra of
each oligoglucuronan fraction. These results lead us to propose that
the enzyme cleavage mode was a -elimination. The presence of
oligoglucuronans with dp of 2 to >7 after substrate digestion
indicates that the purified lyase was a randomly endolytic enzyme, and
these results agree with the literature (40). The glucuronan lyase obtained may be used to produce pure oligoglucuronan fractions unacetylated or partially acetylated (data not shown) in
order to test the biological function of the lyase during the life
cycle of S. meliloti M5N1CS (NCIMB 40472). On the basis of these results, further studies will be performed in order to clone the
glucuronan lyase gene and produce the enzyme. Oligoglucuronans will be
produced in large amounts and tested for biological properties as other
bacterial oligosaccharides (21, 34).
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ACKNOWLEDGMENT |
This work was supported by the Pôle Génie des
Procédés (Conseil Régional de Picardie).
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire des
Polysaccharides Microbiens et Végétaux, IUT,
Département de Génie Biologique, Avenue des Facultés,
Le Bailly, 80025 Amiens, France. Phone: (33) (3) 22 53 40 99. Fax: (33)
(3) 22 95 62 54. E-mail: Josiane.Courtois{at}iut.u-picardie.fr.
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Applied and Environmental Microbiology, November 2001, p. 5197-5203, Vol. 67, No. 11
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.11.5197-5203.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
