Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae

Letters in Applied Microbiology 2004, 38, 327–332
doi:10.1111/j.1472-765X.2004.01492.x
Fermentation of pectin and glucose, and activity
of pectin-degrading enzymes in the rabbit caecal bacterium
Bacteroides caccae
K. Sirotek1, L. Slova´kova´1, J. Kopec?ny´1 and M. Marounek1,2
1Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic, and 2Research Institute of Animal
Production, Prague, Czech Republic
2003/0767: received 28 August 2003, revised 07 January 2004 and accepted 22 January 2004
A B S T R A C T
K . S I R O T E K , L . S L O V A
´ K O V A´ , J . K O P E C? N Y´ A N D M . M A R O U N E K . 2004.
Aims: To compare fermentation pattern in cultures of Bacteroides caccae supplied with pectin and glucose, and
identify enzymes involved in metabolism of pectin.
Methods and Results: A strain KWN isolated from the rabbit caecum was used. Fermentation pattern, changes of
viscosity and enzyme reactions products were determined. Cultures grown on pectin produced signi?cantly more
acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. Production of cell dry
matter and protein per gram of substrate used was the same in pectin- and glucose-grown cultures. The principal
enzymes that participated in the metabolism of pectin were extracellular exopectate hydrolase (EC 3.2.1.67),
extracellular endopectate lyase (EC 4.2.2.2) and cell-associated 2-keto-3-deoxy-6-phosphogluconate (KDPG)
aldolase (EC 4.1.2.14). The latter enzyme is unique to the Entner–Doudoroff pathway. Activities of pectinolytic
enzymes in cultures grown on glucose were low. Activity of KDPG aldolase was similar in pectin- and glucose-
grown cells.
Conclusions: Metabolites and activities of pectin-degrading enzymes differed in cultures of B. caccae KWN grown
on pectin and glucose. Yields of dry matter and protein were the same on both substrates.
Signi?cance and Impact of the Study: Information on metabolism of pectin in animal strains of Bacteroides is
incomplete. This study extends the knowledge on metabolism in bacteria from the rabbit caecum.
Keywords: Bacteroides caccae, caecum, metabolism, pectin, rabbit.
Macfarlane et al. 1997). Bacteroides counts in fresh faeces
I N T R O D U C T I O N
increased greatly when rats were fed diets containing pectin
Pectin is a ?bre component, occurring in the middle lamella
(Dongowski et al. 2002). Bacteria belonging to the Bacter-
and primary cell wall of higher plants. Like other ?bre
oides predominated over other identi?ed pectinolytic organ-
constituents, pectin is not degraded by endogenous enzymes
isms in the rabbit caecum (Sirotek et al. 2001).
in the stomach and small intestine of man and other animals,
Pectin degradation pattern has been determined in
but fermented in the hindgut (Bacon 1978). Among
Bacteroides from the human intestine. In strains examined,
pectinolytic bacteria isolated from the lower intestinal tract,
pectin was converted to a mixture of oligogalacturonides,
members of the Bacteroides genus are probably the most
mainly unsaturated products of pectate lyase activity
important,
taking
into
account
their
high
numbers
(McCarthy et al. 1985; Jensen and Canale-Parola 1986;
and nutritional versatility (Bayliss and Houston 1984;
Dongowski et al. 2000). Oligogalacturonides were metabo-
lized intracellulary with acetate as the main fermentation
Correspondence to: M. Marounek, Institute of Animal Physiology and Genetics,
product. Little work has been published on the metabolism
Czech Academy of Sciences, Prague 4 – Krc?, CZ-142 20, Czech Republic
(e-mail: marounek@iapg.cas.cz).
of pectin in Bacteroides from other habitats. There are
ª 2004 The Society for Applied Microbiology

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328
K . S I R O T E K ET AL.
studies on metabolism of pectin in Bacteroides ruminicola
determine methanol, methoxyl groups were hydrolysed in an
(Wojciechowicz 1971; Szyman?ski 1981), however, according
alkali milieu (0Æ5 mol l)1 NaOH), and methanol estimated by
to current taxonomic criteria this rumen bacterium is
gas-liquid chromatography on a 2Æ4 m column of Chromosorb
assigned to the genus Prevotella (Avgus?tin et al. 1994).
W AW with 15% SP1220/1% H3PO4 (Supelco, Bellefonte,
Thus, the aim of our study was to elucidate metabolism of
PA, USA), operated at 100°C. Carbon content of pectin was
pectin in a strain of Bacteroides caccae from the rabbit
determined using a Perkin Elmer 2400 elemental analyser
caecum. The isolate KWN grew well on pectin and its
(Perkin-Elmer). Pectin contained neutral sugars and carbon at
pectinolytic activity was higher than that of other Bacteroides
68 and 419 mg g)1 of dry matter, respectively, and 52Æ4% of
strains tested in a previous study (Sirotek et al. 2001).
its carboxyl groups were methylated.
Media
M A T E R I A L A N D M E T H O D S
Throughout the study, B. caccae KWN and Strep. bovis X4
Bacteria
were grown anaerobically on a medium supplemented with
Bacteroides caccae KWN was isolated by Dr V. Rada (CUA
clari?ed caecal extract, yeast extract and pancreatic casein
Prague) from the caecal contents of a rabbit fed with oats
hydrolysate (Slova´kova´ et al. 2002). The medium was
and meadow hay ad libitum. Preliminary identi?cation of the
reduced by 0Æ05% cysteineÆHCl. Bacteroides caccae KWN
isolate was based on its phenotypic characteristics (API 50
was grown on pectin or glucose. Substrates were added at
CHL tests; Biome´rieux, Marcy l’Etoile, France). To deter-
4 g l)1, ?nal concentration. Streptococcus bovis X4 was
mine the taxonomy of the strain KWN more precisely,
cultivated on the mixed substrate: pectin and glucose were
DNA was isolated according to Gregg et al. (1994). The 16S
added to the medium at 2 g l)1 each. Pseudomonas ?uorescens
rDNA fragments were obtained after amplifying bacterial
DBM 3056 was grown on the medium of Van Dijken and
DNA using FP27 (5¢-AGA GTT TGA TCC TGG CTC
Quayle (1977) with glucose (4 g l)1). The pseudomonad was
AGG A-3¢, E. coli position 8–29) and 515R (5¢-TTA CCG
grown aerobically.
TGA CTG GCA C-3¢, E. coli position 520–538) primers on
a thermocycler (Kopec?ny´ et al. 2001). These 500 bp frag-
Metabolites and cell yields
ments were sequenced with the ABI 310 capillary sequencer
(Perkin-Elmer, Boston, MA, USA). After editing, 16S
Medium for B. caccae KWN was distributed in 15-ml
rDNA sequences were compared with published sequences
amounts into CO2-gassed 20-ml ?asks, closed by rubber
of related bacteria from the EMBL (EBI) and GenBank
stoppers, and autoclaved at 110°C for 1 h. One-day culture
(NCBI) nucleotide databases using BLAST and with data
(0Æ3 ml) was used to inoculate the growth medium. Inoculated
from the Ribosomal Database Project (Maidak et al. 1994).
cultures were grown at 39°C overnight (16 h) in six replicates.
Two control organisms were used: Streptococcus bovis X4,
The culture pH fell from 6Æ7–6Æ8 to about 6Æ0 (pectin) or 5Æ8
a bacterium with an endo-type of pectin-depolymerizing
(glucose) in the course of the incubation. Methods for
activity (Wojciechowicz and Ziolecki 1984) and Pseudomonas
determination of cell dry matter, protein, residual pectin
?uorescens DBM 3056, a bacterium with enzymes of the
and glucose and analysis of fermentation products were as
Entner–Doudoroff metabolic pathway (Preiss and Ashwell
described
previously
(Marounek
and
Dus?kova´
1999;
1963). Streptococcus bovis X4 was isolated from the rumen
Slova´kova´ et al. 2002). Carbon content was determined in
?uid of a sheep at this Institute. Pseudomonas ?uorescens
freeze-dried cells harvested from an overnight culture.
DBM 3056, was obtained from the culture collection of the
Methanol and carbon were determined as described above.
Department of Biochemistry and Microbiology of the
Hydrogen production in B. caccae KWN was measured in
Institute of Chemical Technology, Prague. Bacteria were
100-ml ?asks hermetically closed with butyl rubber stop-
maintained in 20% (v/v) glycerol at )40°C.
pers. Inoculated cultures (30 ml) were grown in six repli-
cates at 39°C for 16 h. Samples of the headspace gas were
taken with a gas-tight syringe and analysed by gas chroma-
Pectin
tography at 100°C, using a chromatograph equipped with a
Apple pectin was supplied by Pektin Ltd (now Danisco Czech
thermal conductivity detector and a column of Carboxen 100
Republic, Smir?ice, Czech Republic). It was puri?ed by
(Supelco).
ethanol extraction (75% v/v) to remove low-molecular weight
contaminants. Uronic acid content of pectin was determined
Enzyme assays and calculations
by the 3-phenylphenol method (Blumenkrantz and Asboe-
Hansen 1973), and neutral monosaccharide composition by
For enzyme assays, B. caccae KWN was grown on pectin or
gas chromatography (Marounek and Dus?kova´ 1999). To
glucose in 500-ml batch cultures at 39°C for 16 h. One-day
ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 327–332, doi:10.1111/j.1472-765X.2004.01492.x

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M E T A B O L I S M O F P E C T I N I N B . C A C C A E
329
cultures (2 ml) were used to inoculate the growth medium.
shaking water bath for 48 h. In both bacteria, the activity of
Cells were collected by centrifugation from an early
6-phosphogluconate dehydrase (EC 4.2.1.12) and KDPG
stationary phase, washed, and disrupted by sonication (see
aldolase was determined in a coupled reaction with
Marounek and Dus?kova´ 1999 for details). Culture superna-
6-phosphogluconate as the substrate.
tant ?uids were dialysed at 4°C for 24 h. Cell extracts and
Production of metabolites, cell dry matter and protein was
dialysed supernatant ?uids were used for determination of
related to the amount of substrate utilized. Carbon recovery
cell-associated and extracellular activity, respectively, of
was calculated from the metabolic products and C content of
pectate lyase and pectinase (Dus?kova´ and Marounek 2001).
the cells (46Æ4%). Enzyme activities were expressed in
Pectate lyase releases products with an unsaturated residue
nanomoles of substrate split or product liberated per minute
at the nonreducing end. Pectinase cleaves the macromol-
per milligram of protein. The signi?cance of differences was
ecule by hydrolysis. The activity of the former enzyme can
evaluated by the t-test.
be assayed by determination of the absorbance at 232 nm,
and activity of the latter enzyme by determination of the
R E S U L T S
concentration of reducing sugars (Collmer et al. 1988).
Endo- and exo-acting polysaccharidases differ in the rate
Molecular-genetic analysis identi?ed the KWN isolate as a
of the decrease in viscosity of the substrate solution
strain of B. caccae with 98% identity of 16S rDNA
(Rombouts and Pilnik 1980). Lyases have an alkaline pH
sequence. Bacteroides caccae KWN utilized almost all
optimum and require divalent cations. Hydrolases have pH
glucose and 81% of pectin. Cultures grown on pectin
optimum 6Æ0 or lower and do not require divalent cations
produced signi?cantly more acetate and less formate, lactate,
(Collmer et al. 1988). Lyase and hydrolase activity assays,
fumarate and succinate than cultures grown on glucose. As
thus were performed at pH 7Æ5 or 5Æ6, with or without
expected, methanol was found only in former cultures
calcium chloride addition, respectively. This arrangement
(Table 1). Production of hydrogen was very small. Yields of
enabled to distinguish between hydrolase and lyase type of
dry matter and protein were the same in cultures grown on
pectin-degrading activity (McCarthy et al. 1985). To dis-
pectin and glucose. Carbon recovery did not differ greatly in
tinguish between endo-and exo-type of pectin-depolymer-
these cultures.
izing activity of lyase and hydrolase, cultures of B. caccae
Pectin macromolecule was degraded by the action of
KWN and Strep. bovis X4 were grown on pectin for 16 h
pectate lyase and pectinase. Speci?c activities of both
(strain KWN), and on pectin + glucose for 12 h (strain X4).
enzymes were higher in culture supernatants than in cell
Cells were harvested by centrifugation and culture supern-
extracts, and ca 10-times lower in cultures grown on glucose
atants dialysed. A reaction mixture was prepared consisting
than on pectin (Table 2). Action pattern of pectic enzymes
of 100 ml of 1Æ2% (w/v) pectin in 0Æ1 M Na-acetate buffer
(pH 7Æ5 or 5Æ6) and 20 ml of dialysed supernatant ?uid. One
half of the mixture was used for viscosity measurements at
Table 1 Metabolite pro?les and cell yields of the rabbit caecal
39°C in a Hoeppler viscosimeter B3 (VEB MLW;
bacterium Bacteroides caccae KWN grown on pectin and glucose*
Pru¨fgera¨te-Werk, Medingen, Germany). The time of fall
Pectin
Glucose
of a ball in the reaction mixture, hermetically closed, was
measured at intervals according to the manufacturer’s
Substrate used (g l)1)
3Æ25 ± 0Æ19 
3Æ99 ± 0Æ01
instruction. Simultaneously, the second half of the reaction
Metabolites (mmol g)1)
Formate
0Æ9 ± 0Æ3 
2Æ5 ± 0Æ6
mixture was incubated anaerobically at 39°C and samples
Acetate
24Æ9 ± 0Æ7 
17Æ7 ± 0Æ5
were taken for the estimation of reducing sugars (Lever
Propionate
7Æ6 ± 0Æ7
7Æ8 ± 0Æ8
1977), or unsaturated products measured as absorbance
Lactate
0 
4Æ5 ± 0Æ9
at 232 nm. The reaction mixture in lyase activity assay
Fumarate
0Æ3 ± 0Æ1 
1Æ3 ± 0Æ5
was supplemented with CaCl2 at 7Æ5 mmol l)1, ?nal
Succinate
5Æ4 ± 0Æ9 
9Æ6 ± 1Æ7
concentration.
Methanol
11Æ4 ± 1Æ7+
0
The
activity
of
2-keto-3-deoxy-6-phosphogluconate
Hydrogen
0Æ12 ± 0Æ01
0Æ13 ± 0Æ03
(KDPG) aldolase (EC 4.1.2.14), an enzyme unique to the
Cell dry weight (g l)1)
1Æ06 ± 0Æ17 
1Æ31 ± 0Æ06
Entner–Doudoroff pathway, was determined as described by
Cell yields (g g)1 substrate used)
Marounek and Dus?kova´ (1999), with KDPG as the
Dry matter
0Æ33 ± 0Æ05
0Æ33 ± 0Æ02
substrate. To test the method, the activity of KDPG
Protein
0Æ10 ± 0Æ01
0Æ10 ± 0Æ01
C-recovery (%)
121Æ0
127Æ2
aldolase was determined in glucose-cultivated cultures of Ps.
?uorescens DBM 3056. The growth medium (500 ml in 1Æ5-l
*Means of six cultures ±S.D. The results are differences in amounts
?asks) was inoculated with 2 ml of 2 day-culture of the
between the beginning and end of the incubation.
pseudomonad, and cultivated aerobically at 28°C on a
 Signi?cantly different from the glucose value (P < 0Æ01).
ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 327–332, doi:10.1111/j.1472-765X.2004.01492.x

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330
K . S I R O T E K ET AL.
Table 2 Speci?c activities of PL, P, KDPGA, and PGD plus
(a)
KDPGA in cells and culture supernatant ?uids of Bacteroides caccae
) 500
100
KWN and Pseudomonas ?uorescens DBM 3056. Bacteria were grown on
–1
pectin or glucose
400
80
mol g
µ

Glucose
300
60
Enzyme
Pectin
activity*
Sample
B. caccae
B. caccae Ps. ?uorescens
200
40
PL
Cell extract
2Æ9 ± 0Æ5
0Æ3 ± 0Æ1 –
100
20
elative viscosity (%)
PL
Supernatant 30Æ2 ± 5Æ1 2Æ8 ± 0Æ7 –
R
Double bonds (
P
Cell extract
5Æ5 ± 1Æ1
0Æ5 ± 0Æ1 –
0
0
P
Supernatant 45Æ9 ± 7Æ9 4Æ5 ± 0Æ3 –
0
1
2
KDPGA
Cell extract
662 ± 55
719 ± 31 468
PGD + KDPGA Cell extract
0
0
94
(b)
) 100
100
–1
*Expressed in nanomoles of substrate split or product released per
80
80
minute per milligram of protein. See Material and methods for
mol g
substrates of enzymatic reactions.
µ

60
60
Means of two (enzymes of Ps. ?uorescens) or four (other enzymes)
40
cultures ±
40
S.D.
–, not determined; PL, pectate lyase; P, pectinase; KDPGA, 2-keto-3-
20
20
deoxy-6-phosphogluconate aldolase and PGD, 6-phosphogluconate
Relative viscosity (%)
dehydrase.
0
0
Reducing sugars (
0
1
2
Time (h)
was determined by viscosimetric and reaction product
Fig. 1 Formation of unsaturated products (a) and production of
analyses. Figure 1a shows that the formation of unsaturated
reducing sugars (b) in reaction mixture of culture supernatant and
products and drop in viscosity were more rapid in the
solution of pectin. Dashed lines show relative viscosity of reaction
culture supernatant of Strep. bovis X4 than in that of
mixture. Closed symbols: Bacteroides caccae KWN; open symbols:
B. caccae KWN. The ratio of both parameters (micromoles
Streptococcus bovis X4
of double bonds per gram per 1% of the relative viscosity
decrease), however, was almost the same in both bacteria:
15Æ6 and 16Æ0 in Strep. bovis and B. caccae, respectively.
pyruvate requires metabolic hydrogen, was lower on
A great increase of the reducing sugar concentration in the
pectin. Lactate was produced only in cultures supplied
B. caccae KWN culture supernatant supplied with pectin
with glucose. In accordance with Johnson et al. (1986),
was accompanied by a negligible reduction of viscosity
only a trace of hydrogen was detected in the headspace
(Fig. 1b). Contrary to this, a relatively small increase of the
gas. Yields of cell dry matter and protein suggest that the
reducing power and a substantial loss of viscosity were
gain of energy on both substrates was similar. C-recover-
observed in the culture supernatant of Strep. bovis X4.
ies, however, exceeded 100%, indicating that also com-
Both pectin- and glucose-grown cells of B. caccae KWN
pounds other than pectin and glucose were used for the
possessed
2-keto-3-deoxy-6-phosphogluconate
(KDPG)
synthesis of cell matter.
aldolase (EC 4.1.2.14) activity. Phosphogluconate was
Depolymerases of pectin split the glycosidic bonds either
metabolized by the cell extract of Ps. ?uorescens, but not
by b-elimination (lyases) or by hydrolysis (hydrolases).
by the cell extract of B. caccae.
Enzymes of both types can degrade the macromolecule in a
random fashion, or liberate galacturonate residues by a
terminal attack on the polymer (Rombouts and Pilnik
D I S C U S S I O N
1980). Comparison of the time course of the concentration
Pectin is a more oxidized substrate than glucose, thus its
of reducing sugars and relative viscosity (Fig. 1b) indicates
metabolites should be less reduced than those of glucose.
that the pectin hydrolase has an exo-type mode of action
Indeed, production of acetate, the formation of, which
(terminal cleavage of the polymer) in B. caccae KWN, but
does not require reducing equivalents, was higher by
an endo-type mode in Strep. bovis X4 (random cleavage).
40Æ7% in cultures grown on pectin compared with those
The lyase activity of Strep. bovis X4 was twofold higher
supplied with glucose (by 72Æ7% when expressed per gram
than that of B. caccae KWN. Action patterns of lyases,
of substrate used). However, the production of lactate,
however,
were
similar
in
both
bacteria
(Fig. 1a).
fumarate and succinate, the synthesis of which from
Wojciechowicz
and
Ziolecki
(1984)
identi?ed
the
ª 2004 The Society for Applied Microbiology, Letters in Applied Microbiology, 38, 327–332, doi:10.1111/j.1472-765X.2004.01492.x

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331
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This research was supported by grant 525/03/0358 of the
Maidak, B.L., Larsen, N., McCaughey, M.J., Overbeek, R., Olsen,
Czech Science Foundation. The authors wish to thank the
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