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

2004; Oxford University Press; Volume: 38; Issue: 4 Linguagem: Inglês

10.1111/j.1472-765x.2004.01492.x

1472-765X

K. Sirotek, Ľudmila Slováková, J Kopečný, M. Marounek,

Polysaccharides Composition and Applications

Letters in Applied MicrobiologyVolume 38, Issue 4 p. 327-332 Free Access Fermentation of pectin and glucose, and activity of pectin-degrading enzymes in the rabbit caecal bacterium Bacteroides caccae K. Sirotek, K. Sirotek Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech RepublicSearch for more papers by this authorL. Slováková, L. Slováková Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech RepublicSearch for more papers by this authorJ. Kopečný, J. Kopečný Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech RepublicSearch for more papers by this authorM. Marounek, M. Marounek Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic Research Institute of Animal Production, Prague, Czech RepublicSearch for more papers by this author K. Sirotek, K. Sirotek Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech RepublicSearch for more papers by this authorL. Slováková, L. Slováková Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech RepublicSearch for more papers by this authorJ. Kopečný, J. Kopečný Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech RepublicSearch for more papers by this authorM. Marounek, M. Marounek Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic Research Institute of Animal Production, Prague, Czech RepublicSearch for more papers by this author First published: 17 February 2004 https://doi.org/10.1111/j.1472-765X.2004.01492.xCitations: 10 M. Marounek, Institute of Animal Physiology and Genetics, Czech Academy of Sciences, Prague 4 – Krč, CZ-142 20, Czech Republic (e-mail: marounek@iapg.cas.cz). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract 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 significantly 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. Significance 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. Introduction Pectin is a fibre component, occurring in the middle lamella and primary cell wall of higher plants. Like other fibre constituents, pectin is not degraded by endogenous enzymes in the stomach and small intestine of man and other animals, but fermented in the hindgut (Bacon 1978). Among pectinolytic bacteria isolated from the lower intestinal tract, members of the Bacteroides genus are probably the most important, taking into account their high numbers and nutritional versatility (Bayliss and Houston 1984; Macfarlane et al. 1997). Bacteroides counts in fresh faeces increased greatly when rats were fed diets containing pectin (Dongowski et al. 2002). Bacteria belonging to the Bacteroides predominated over other identified pectinolytic organisms in the rabbit caecum (Sirotek et al. 2001). Pectin degradation pattern has been determined in Bacteroides from the human intestine. In strains examined, pectin was converted to a mixture of oligogalacturonides, mainly unsaturated products of pectate lyase activity (McCarthy et al. 1985; Jensen and Canale-Parola 1986; Dongowski et al. 2000). Oligogalacturonides were metabolized intracellulary with acetate as the main fermentation product. Little work has been published on the metabolism of pectin in Bacteroides from other habitats. There are studies on metabolism of pectin in Bacteroides ruminicola (Wojciechowicz 1971; Szymaňski 1981), however, according to current taxonomic criteria this rumen bacterium is assigned to the genus Prevotella (Avguštin et al. 1994). Thus, the aim of our study was to elucidate metabolism of pectin in a strain of Bacteroides caccae from the rabbit caecum. The isolate KWN grew well on pectin and its pectinolytic activity was higher than that of other Bacteroides strains tested in a previous study (Sirotek et al. 2001). Material and methods Bacteria Bacteroides caccae KWN was isolated by Dr V. Rada (CUA Prague) from the caecal contents of a rabbit fed with oats and meadow hay ad libitum. Preliminary identification of the isolate was based on its phenotypic characteristics (API 50 CHL tests; Biomérieux, Marcy l'Etoile, France). To determine the taxonomy of the strain KWN more precisely, DNA was isolated according to Gregg et al. (1994). The 16S rDNA fragments were obtained after amplifying bacterial DNA using FP27 (5′-AGA GTT TGA TCC TGG CTC AGG A-3′, E. coli position 8–29) and 515R (5′-TTA CCG TGA CTG GCA C-3′, E. coli position 520–538) primers on a thermocycler (Kopečnýet al. 2001). These 500 bp fragments were sequenced with the ABI 310 capillary sequencer (Perkin-Elmer, Boston, MA, USA). After editing, 16S rDNA sequences were compared with published sequences of related bacteria from the EMBL (EBI) and GenBank (NCBI) nucleotide databases using BLAST and with data from the Ribosomal Database Project (Maidak et al. 1994). Two control organisms were used: Streptococcus bovis X4, a bacterium with an endo-type of pectin-depolymerizing activity (Wojciechowicz and Ziolecki 1984) and Pseudomonas fluorescens DBM 3056, a bacterium with enzymes of the Entner–Doudoroff metabolic pathway (Preiss and Ashwell 1963). Streptococcus bovis X4 was isolated from the rumen fluid of a sheep at this Institute. Pseudomonas fluorescens DBM 3056, was obtained from the culture collection of the Department of Biochemistry and Microbiology of the Institute of Chemical Technology, Prague. Bacteria were maintained in 20% (v/v) glycerol at −40°C. Pectin Apple pectin was supplied by Pektin Ltd (now Danisco Czech Republic, Smiřice, Czech Republic). It was purified by ethanol extraction (75% v/v) to remove low-molecular weight contaminants. Uronic acid content of pectin was determined by the 3-phenylphenol method (Blumenkrantz and Asboe-Hansen 1973), and neutral monosaccharide composition by gas chromatography (Marounek and Dušková 1999). To determine methanol, methoxyl groups were hydrolysed in an alkali milieu (0·5 mol l−1 NaOH), and methanol estimated by gas-liquid chromatography on a 2·4 m column of Chromosorb W AW with 15% SP1220/1% H3PO4 (Supelco, Bellefonte, PA, USA), operated at 100°C. Carbon content of pectin was determined using a Perkin Elmer 2400 elemental analyser (Perkin-Elmer). Pectin contained neutral sugars and carbon at 68 and 419 mg g−1 of dry matter, respectively, and 52·4% of its carboxyl groups were methylated. Media Throughout the study, B. caccae KWN and Strep. bovis X4 were grown anaerobically on a medium supplemented with clarified caecal extract, yeast extract and pancreatic casein hydrolysate (Slovákováet al. 2002). The medium was reduced by 0·05% cysteine·HCl. Bacteroides caccae KWN was grown on pectin or glucose. Substrates were added at 4 g l−1, final concentration. Streptococcus bovis X4 was cultivated on the mixed substrate: pectin and glucose were added to the medium at 2 g l−1 each. Pseudomonas fluorescens DBM 3056 was grown on the medium of Van Dijken and Quayle (1977) with glucose (4 g l−1). The pseudomonad was grown aerobically. Metabolites and cell yields Medium for B. caccae KWN was distributed in 15-ml amounts into CO2-gassed 20-ml flasks, closed by rubber stoppers, and autoclaved at 110°C for 1 h. One-day culture (0·3 ml) was used to inoculate the growth medium. Inoculated cultures were grown at 39°C overnight (16 h) in six replicates. The culture pH fell from 6·7–6·8 to about 6·0 (pectin) or 5·8 (glucose) in the course of the incubation. Methods for determination of cell dry matter, protein, residual pectin and glucose and analysis of fermentation products were as described previously (Marounek and Dušková 1999; Slovákováet al. 2002). Carbon content was determined in freeze-dried cells harvested from an overnight culture. Methanol and carbon were determined as described above. Hydrogen production in B. caccae KWN was measured in 100-ml flasks hermetically closed with butyl rubber stoppers. Inoculated cultures (30 ml) were grown in six replicates at 39°C for 16 h. Samples of the headspace gas were taken with a gas-tight syringe and analysed by gas chromatography at 100°C, using a chromatograph equipped with a thermal conductivity detector and a column of Carboxen 100 (Supelco). Enzyme assays and calculations For enzyme assays, B. caccae KWN was grown on pectin or glucose in 500-ml batch cultures at 39°C for 16 h. One-day cultures (2 ml) were used to inoculate the growth medium. Cells were collected by centrifugation from an early stationary phase, washed, and disrupted by sonication (see Marounek and Dušková 1999 for details). Culture supernatant fluids were dialysed at 4°C for 24 h. Cell extracts and dialysed supernatant fluids were used for determination of cell-associated and extracellular activity, respectively, of pectate lyase and pectinase (Dušková and Marounek 2001). Pectate lyase releases products with an unsaturated residue at the nonreducing end. Pectinase cleaves the macromolecule by hydrolysis. The activity of the former enzyme can be assayed by determination of the absorbance at 232 nm, and activity of the latter enzyme by determination of the concentration of reducing sugars (Collmer et al. 1988). Endo- and exo-acting polysaccharidases differ in the rate of the decrease in viscosity of the substrate solution (Rombouts and Pilnik 1980). Lyases have an alkaline pH optimum and require divalent cations. Hydrolases have pH optimum 6·0 or lower and do not require divalent cations (Collmer et al. 1988). Lyase and hydrolase activity assays, thus were performed at pH 7·5 or 5·6, with or without calcium chloride addition, respectively. This arrangement enabled to distinguish between hydrolase and lyase type of pectin-degrading activity (McCarthy et al. 1985). To distinguish between endo-and exo-type of pectin-depolymerizing activity of lyase and hydrolase, cultures of B. caccae KWN and Strep. bovis X4 were grown on pectin for 16 h (strain KWN), and on pectin + glucose for 12 h (strain X4). Cells were harvested by centrifugation and culture supernatants dialysed. A reaction mixture was prepared consisting of 100 ml of 1·2% (w/v) pectin in 0·1 m Na-acetate buffer (pH 7·5 or 5·6) and 20 ml of dialysed supernatant fluid. One half of the mixture was used for viscosity measurements at 39°C in a Hoeppler viscosimeter B3 (VEB MLW; Prüfgeräte-Werk, Medingen, Germany). The time of fall of a ball in the reaction mixture, hermetically closed, was measured at intervals according to the manufacturer's instruction. Simultaneously, the second half of the reaction mixture was incubated anaerobically at 39°C and samples were taken for the estimation of reducing sugars (Lever 1977), or unsaturated products measured as absorbance at 232 nm. The reaction mixture in lyase activity assay was supplemented with CaCl2 at 7·5 mmol l−1, final concentration. The activity of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14), an enzyme unique to the Entner–Doudoroff pathway, was determined as described by Marounek and Dušková (1999), with KDPG as the substrate. To test the method, the activity of KDPG aldolase was determined in glucose-cultivated cultures of Ps. fluorescens DBM 3056. The growth medium (500 ml in 1·5-l flasks) was inoculated with 2 ml of 2 day-culture of the pseudomonad, and cultivated aerobically at 28°C on a shaking water bath for 48 h. In both bacteria, the activity of 6-phosphogluconate dehydrase (EC 4.2.1.12) and KDPG aldolase was determined in a coupled reaction with 6-phosphogluconate as the substrate. Production of metabolites, cell dry matter and protein was related to the amount of substrate utilized. Carbon recovery was calculated from the metabolic products and C content of the cells (46·4%). Enzyme activities were expressed in nanomoles of substrate split or product liberated per minute per milligram of protein. The significance of differences was evaluated by the t-test. Results Molecular-genetic analysis identified the KWN isolate as a strain of B. caccae with 98% identity of 16S rDNA sequence. Bacteroides caccae KWN utilized almost all glucose and 81% of pectin. Cultures grown on pectin produced significantly more acetate and less formate, lactate, fumarate and succinate than cultures grown on glucose. As expected, methanol was found only in former cultures (Table 1). Production of hydrogen was very small. Yields of dry matter and protein were the same in cultures grown on pectin and glucose. Carbon recovery did not differ greatly in these cultures. Table 1. Metabolite profiles and cell yields of the rabbit caecal bacterium Bacteroides caccae KWN grown on pectin and glucose* Pectin Glucose Substrate used (g l−1) 3·25 ± 0·19† 3·99 ± 0·01 Metabolites (mmol g−1) Formate 0·9 ± 0·3† 2·5 ± 0·6 Acetate 24·9 ± 0·7† 17·7 ± 0·5 Propionate 7·6 ± 0·7 7·8 ± 0·8 Lactate 0† 4·5 ± 0·9 Fumarate 0·3 ± 0·1† 1·3 ± 0·5 Succinate 5·4 ± 0·9† 9·6 ± 1·7 Methanol 11·4 ± 1·7+ 0 Hydrogen 0·12 ± 0·01 0·13 ± 0·03 Cell dry weight (g l−1) 1·06 ± 0·17† 1·31 ± 0·06 Cell yields (g g−1 substrate used) Dry matter 0·33 ± 0·05 0·33 ± 0·02 Protein 0·10 ± 0·01 0·10 ± 0·01 C-recovery (%) 121·0 127·2 *Means of six cultures ±s.d. The results are differences in amounts between the beginning and end of the incubation. †Significantly different from the glucose value (P < 0·01). Pectin macromolecule was degraded by the action of pectate lyase and pectinase. Specific activities of both enzymes were higher in culture supernatants than in cell extracts, and ca 10-times lower in cultures grown on glucose than on pectin (Table 2). Action pattern of pectic enzymes was determined by viscosimetric and reaction product analyses. Figure 1a shows that the formation of unsaturated products and drop in viscosity were more rapid in the culture supernatant of Strep. bovis X4 than in that of B. caccae KWN. The ratio of both parameters (micromoles of double bonds per gram per 1% of the relative viscosity decrease), however, was almost the same in both bacteria: 15·6 and 16·0 in Strep. bovis and B. caccae, respectively. A great increase of the reducing sugar concentration in the B. caccae KWN culture supernatant supplied with pectin was accompanied by a negligible reduction of viscosity (Fig. 1b). Contrary to this, a relatively small increase of the reducing power and a substantial loss of viscosity were observed in the culture supernatant of Strep. bovis X4. Table 2. Specific activities of PL, P, KDPGA, and PGD plus KDPGA in cells and culture supernatant fluids of Bacteroides caccae KWN and Pseudomonas fluorescens DBM 3056. Bacteria were grown on pectin or glucose Enzyme activity* Sample Pectin B. caccae Glucose B. caccae Ps. fluorescens PL Cell extract 2·9 ± 0·5 0·3 ± 0·1 – PL Supernatant 30·2 ± 5·1 2·8 ± 0·7 – P Cell extract 5·5 ± 1·1 0·5 ± 0·1 – P Supernatant 45·9 ± 7·9 4·5 ± 0·3 – KDPGA Cell extract 662 ± 55 719 ± 31 468 PGD + KDPGA Cell extract 0 0 94 *Expressed in nanomoles of substrate split or product released per minute per milligram of protein. See Material and methods for substrates of enzymatic reactions. Means of two (enzymes of Ps. fluorescens) or four (other enzymes) cultures ±s.d. –, not determined; PL, pectate lyase; P, pectinase; KDPGA, 2-keto-3-deoxy-6-phosphogluconate aldolase and PGD, 6-phosphogluconate dehydrase. Figure 1Open in figure viewerPowerPoint Formation of unsaturated products (a) and production of reducing sugars (b) in reaction mixture of culture supernatant and solution of pectin. Dashed lines show relative viscosity of reaction mixture. Closed symbols: Bacteroides caccae KWN; open symbols: Streptococcus bovis X4 Both pectin- and glucose-grown cells of B. caccae KWN possessed 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (EC 4.1.2.14) activity. Phosphogluconate was metabolized by the cell extract of Ps. fluorescens, but not by the cell extract of B. caccae. Discussion Pectin is a more oxidized substrate than glucose, thus its metabolites should be less reduced than those of glucose. Indeed, production of acetate, the formation of, which does not require reducing equivalents, was higher by 40·7% in cultures grown on pectin compared with those supplied with glucose (by 72·7% when expressed per gram of substrate used). However, the production of lactate, fumarate and succinate, the synthesis of which from pyruvate requires metabolic hydrogen, was lower on pectin. Lactate was produced only in cultures supplied with glucose. In accordance with Johnson et al. (1986), only a trace of hydrogen was detected in the headspace gas. Yields of cell dry matter and protein suggest that the gain of energy on both substrates was similar. C-recoveries, however, exceeded 100%, indicating that also compounds other than pectin and glucose were used for the synthesis of cell matter. Depolymerases of pectin split the glycosidic bonds either by β-elimination (lyases) or by hydrolysis (hydrolases). 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 1980). Comparison of the time course of the concentration of reducing sugars and relative viscosity (Fig. 1b) indicates that the pectin hydrolase has an exo-type mode of action (terminal cleavage of the polymer) in B. caccae KWN, but an endo-type mode in Strep. bovis X4 (random cleavage). The lyase activity of Strep. bovis X4 was twofold higher than that of B. caccae KWN. Action patterns of lyases, however, were similar in both bacteria (Fig. 1a). Wojciechowicz and Ziolecki (1984) identified the pectin-degrading enzyme of Strep. bovis 13E as endopolygalacturonate (i.e. endopectate) lyase (EC 4.2.2.2). We propose that an enzyme of the same type is produced in pectin-grown cultures of the strain KWN. Specific activity of extracellular pectinolytic enzymes of the strain KWN was by one order higher than that of cell-associated enzymes. Although a part of the extracellular pectinolytic activity may be released into the environment by cell lysis, we believe on basis of light microscopy examinations that most of this activity was produced by nonlysed cells. This has similarly been shown to be the case for human colonic bacteria B. pectinophilus and B. galacturonicus (Jensen and Canale-Parola 1986). On the contrary, polygalacturonic acid (PGA) lyase and PGA hydrolase of B. thetaiotamicron were cell-associated (McCarthy et al. 1985). The distinction between extracellular and cell-associated enzymes, however, is somewhat artificial as in natural ecosystems an extracellular polysaccharidase is trapped between the bacterium and the plant particle, making the enzyme effectively cell-associated (Salyers and Leedle 1983). High concentration of methanol in pectin-grown cultures of B. caccae KWN suggests that methoxyl groups of pectin were hydrolyzed. Products of pectin degradation are catabolized intracellulary. Cells of the strain KWN possess the activity of KDPG aldolase, the key enzyme of the Entner–Doudoroff pathway of uronate metabolism, as has been shown also in other pectin-utilizing rumen and rabbit caecal bacteria (Paster and Canale-Parola 1985; Marounek and Dušková 1999; Dušková and Marounek 2001; Slovákováet al. 2002), and in various saprophytic micro-organisms and plant pathogens (Rombouts and Pilnik 1980). Phosphogluconate, a product of the glucose metabolism, was not metabolized by the cell extracts of B. caccae KWN. This indicates that phosphogluconate dehydrase (EC 4.2.1.12), the enzyme producing KDPG from 6-phosphogluconate (Touster 1969) was absent. Consequently, the conventional Entner–Doudoroff pathway of glucose metabolism cannot operate in B. caccae. 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