Regulation and distribution of Fibrobacter succinogenes subsp. succinogenes S85 endoglucanases

The distribution of endoglucanase activities in cultures of Fibrobacter succinogenes subsp. succinogenes S85 grown on different carbon sources was examined by a variety of biochemical and immunological techniques. Total culture endoglucanase activity was primarily cell associated and was expressed constitutively, although synthesis of endoglucanase 1 (EG1) was repressed by cellobiose. Western immunoblotting showed that EG1 and EG3 were released into the culture fluid during growth, while EG2 remained largely associated with the cell. Subcellular localization showed low endoglucanase activity in the periplasmic fraction and similar, high levels in the cytoplasmic and membrane fractions. Western immunoblotting showed that EG2 was absent from the periplasmic fraction. Data from immunoelectron microscopy with either polyclonal or monoclonal antibody to EG2 revealed a high density of gold labeling at sites where there was a disruption in the regular features of the cell surface, such as in blebbing or physical tearing of the membrane. When cells were grown on cellulose, there was a high density of labeling on the cellulose but not on the cells, indicating that EG2 has limited exposure at the cell surface. On the basis of these data, export of enzymes from their intracellular locations appears to occur via three different mechanisms: a specific secretory pathway independent of cellulose, a secretory mechanism which is mediated by contact with cellulose, and a generalized blebbing process that occurs irrespective of the carbon source.     

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.0 mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl cellulose.     

Purification, characterization, and mode of action of endoxylanases 1 and 2 from Fibrobacter succinogenes S85.

Two different endoxylanases (1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8), designated 1 and 2, have been purified by column chromatography to apparent homogeneity from the nonsedimentable extracellular culture fluid of the strictly anaerobic, ruminal bacterium Fibrobacter succinogenes S85 grown on crystalline cellulose. Endoxylanases 1 and 2 were shown to be basic proteins of 53.7 and 66.0 kDa, respectively, with different pH and temperature optima, as well as different substrate hydrolysis characteristics. The Km and Vmax values with water-soluble oat spelts xylan as substrate were 2.6 mg ml-1 and 33.6 mumol min-1 mg-1 for endoxylanase 1 and 1.3 mg ml-1 and 118 mumol min-1 mg-1 for endoxylanase 2. Endoxylanase 1, but not endoxylanase 2, released arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, but not from arabinan, arabinogalactan, or aryl-alpha-L-arabinofuranosides. With an extended hydrolysis time, endoxylanase 1 released 62.5 and 50% of the available arabinose from water-soluble oat spelts xylan and rye flour arabinoxylan, respectively. Endoxylanase 1 released arabinose directly from the xylan backbone, and this preceded hydrolysis of the xylan to xylooligosaccharides. Endoxylanase 2 showed significant activity against carboxymethyl cellulose but was unable to substantially hydrolyze acid-swollen cellulose. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on water-soluble xylan and xylooligosaccharides. Because of their unique hydrolytic properties, endoxylanases 1 and 2 appear to have strategic roles in plant cell wall digestion by F. succinogenes in vivo.     

Isolation of a Cellodextrinase from Bacteroides succinogenes

An enzyme which released the cellobiose group from p-nitrophenyl cellobioside was isolated from the periplasmic space of Bacteroides succinogenes grown on Avicel crystalline cellulose in a continuous cultivation system and separated from endoglucanases by column chromatography. The molecular weight of the enzyme was approximately 40,000, as estimated by gel filtration. The enzyme has an isoelectric point of 4.9. The enzyme exhibited low hydrolytic activity on acid-swollen cellulose and practically no activity on carboxymethyl cellulose, Avicel cellulose, and cellobiose, but it hydrolyzed p-nitrophenyl lactoside and released cellobiose from cellotriose and from higher cello-oligosaccharides. These data demonstrate that the enzyme is a cellodextrinase with an exotype of function.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85.

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Esterase Activities of Fibrobacter succinogenes subsp. succinogenes S85

Cells of the anaerobic ruminal bacterium Fibrobacter succinogenes subsp. succinogenes S85 (formerly Bacteroides succinogenes) exhibit arylesterase activity. When cells were grown on cellulose, it was found that 69% of the total esterase activity was extracellular while 65% was nonsedimentable upon centrifugation of the culture supernatant at 100,000 x g. Disruption of the cells by various different methods failed to increase the esterase activity, indicating that the substrate was fully accessible to esterase enzymes in intact cells. During growth of cells with either glucose or cellulose as the sole carbon source, the increase in acetylesterase activity corresponded to an increase in cell density, suggesting constitutive production. The enzyme(s) hydrolyzed alpha-naphthyl, p-nitrophenyl, and 4-methylumbelliferyl derivatives of acetic acid; xylose tetraacetate; glucose pentaacetate; acetylxylan; and a polymer composed of ferulic acid, arabinose, and xylose in molar proportions of 1:1.1:2.2 (FAX). These data demonstrate the presence of an acetylxylan esterase and a ferulic acid esterase. The cleavage of FAX also documents the presence of an alpha-l-arabinofuranosidase.     

Molecular cloning and expression in Escherichia coli of a cellodextrinase gene from Bacteroides succinogenes S85

A DNA fragment coding for a cellodextrinase of Bacteroides succinogenes S85 was isolated by screening of a pBR322 gene library in Escherichia coli HB101. Of 100,000 colonies screened on a complex medium with methylumbelliferyl-beta-D-cellobioside as the indicator substrate, two cellodextrinase-positive clones (CB1 and CB2) were isolated. The DNA inserts from the two recombinant plasmids were 7.7 kilobase pairs in size and had similar restriction maps. After subcloning from pCB2, a 2.5-kilobase-pair insert which coded for cellodextrinase activity was isolated. The enzyme was located in the cytoplasm of the E. coli host. It exhibited no activity on carboxymethyl cellulose, Avicel microcrystalline cellulose, acid-swollen cellulose, or cellobiose but hydrolyzed p-nitrophenyl-beta-D-cellobioside and p-nitrophenyl-beta-D-lactoside. The Km (0.1 mM) for the hydrolysis of p-nitrophenyl-cellobioside by the enzyme expressed in E. coli was similar to that reported for the purified enzyme from B. succinogenes. Expression of the cellodextrinase gene was subjected to catabolite repression by glucose and was not induced by cellobiose. The origin of the DNA insert from B. succinogenes was confirmed by Southern blot analysis. Western blotting (immunoblotting) using antibodies raised against the purified B. succinogenes cellodextrinase revealed a protein with a molecular weight of approximately 50,000 in E. coli clones which comigrated with the native enzyme isolated from B. succinogenes. These data indicate that the cellodextrinase gene expressed in E. coli is fully functional and codes for an enzyme with properties similar to those of the native enzyme.     

Gas-liquid chromatography for evaluating polysaccharide degradation by Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85.

Two predominant rumen cellulolytic bacteria, Ruminococcus flavefaciens C94 and Bacteroides succinogenes S85, were incubated with ground filter paper (Whatman no. 1), cattle manure fiber, wheat straw, Kentucky bluegrass, alfalfa, and corn silage as substrates. Analyses of the initial substrate and the recovered residue after 48 h of static incubation showed that R. flavefaciens C94 was quantitatively more effective than B. succinogenes S85 in degrading total dry matter (32.3% versus 16.1%). However, B. succinogenes S85 demonstrated a qualitative advantage in degrading the hemicellulose and hemicellulosic sugars of particular substrates. R. flavefaciens degraded a mean 29.7% of the cellulose and 35.6% of the hemicellulose in the various substrates, whereas B. succinogenes degraded a mean 17.9 and 31.6% of these fractions, respectively. Gas-liquid chromatography was an important aid in characterizing the polysaccharide-degrading capabilities of these rumen species.     

Catalytic and substrate-binding domains of endoglucanase 2 from Bacteroides succinogenes.

Endoglucanase 2 (EG2) of the cellulolytic ruminal anaerobe Bacteroides succinogenes is a 118-kilodalton (kDa) enzyme which binds to cellulose and produces cellotetraose as the end product of hydrolysis. The purified enzyme was treated with the protease trypsin in an attempt to isolate peptides which retained the ability to either hydrolyze soluble carboxymethyl cellulose or bind to insoluble cellulose. There was no loss in endoglucanase activity (carboxymethylcellulase) over a period of 2 h following the addition of trypsin. In comparison, there was a greater than eightfold reduction in the binding of carboxymethylcellulase activity to crystalline cellulose. A Lineweaver-Burk plot with amorphous cellulose as the substrate revealed that the trypsin-digested enzyme had an identical Vmax but a 1.9-fold-lower Km in comparison with the intact enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the trypsin-digested enzyme revealed two major peptides of 43 and 51 kDa (p43 and p51). The 43-kDa peptide was able to bind to both amorphous and crystalline cellulose, whereas p51 did not. Purified p51 had a molar activity toward carboxymethyl cellulose which was identical to that of the intact enzyme, but activity toward both amorphous and crystalline cellulose was reduced approximately twofold. Two high-titer monoclonal antibodies from mice immunized with the intact protein recognized p43 but not p51. The results are consistent with a bifunctional organization of EG2, in which the 118-kDa enzyme is composed of a 51-kDa catalytic domain and a highly antigenic 43-kDa substrate-binding domain. In terms of its domain structure and activity toward cellulose, EG2 is very similar to cellobiohydrolase II of Trichoderma reesei.     

Production of maltodextrin 1-phosphate by Fibrobacter succinogenes S85

We show for the first time the occurrence of maltodextrin-1-Phosphate (MD-1P) (DP2) in F. succinogenes S85, a rumen bacterium specialized in cellulolysis which is not able to use maltose and starch. MD-1P were found in intra and extracellular medium of resting cells incubated with glucose. We used 2D 1H NMR technique and TLC to identify their structure and quantify their production with time. It was also shown that these phosphorylated oligosaccharides originated both from exogenous glucose and endogenous glycogen.     

Characterisation of endoglucanases EGB and EGC from Fibrobacter succinogenes

The enzymatic properties of two endoglucanases from Fibrobacter succinogenes, EGB and EGC, were analysed. EGB and EGC were purified from recombinant Escherichia coli cultures expressing their gene. The failure of purification of EGB by classical techniques led us to produce antipeptide antibodies that allowed immunopurification of the protein from E. coli as well as its detection in F. succinogenes cultures. Synthetic peptides were selected from the predicted primary structure of EGB, linked to bovine serum albumin and used as immunogens to obtain specific antibodies. One of the polyclonal antipeptide antisera was used to purify EGB. EGC was purified by affinity chromatography with Ni-NTA resin. The endo mode of action of the two enzymes on carboxymethyl-cellulose was different. The values of K(m) and V(max) were respectively 13.6 mg/ml and 46 micromol/min mg protein for EGB, and 7 mg/ml and 110 micromol/min mg protein for EGC. The reactivity of the antipeptide and the anti-EGC sera with F. succinogenes proteins of molecular mass different from that of EGB and EGC produced in E. coli suggested post-translational modification of the two enzymes in F. succinogenes cultures. Expression of endB and endC genes in F. succinogenes was confirmed by RT-PCR.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Cellulose digestion and cellulase regulation and distribution in Fibrobacter succinogenes subsp. succinogenes S85.

Fibrobacter succinogenes subsp. succinogenes S85 initiated growth on microcrystalline cellulose without a lag whether inoculated from a glucose, cellobiose, or cellulose culture. During growth on cellulose, there was no accumulation of soluble carbohydrate. When the growth medium contained either glucose or cellobiose in combination with microcrystalline cellulose, there was a lag in cellulose digestion until all of the soluble sugar had been utilized, suggesting an end product feedback mechanism that affects cellulose digestion. Cl-stimulated cellobiosidase and periplasmic cellodextrinase were produced under all growth conditions tested, indicating constitutive synthesis. Both cellobiosidases were cell associated until the stationary phase of growth, whereas proteins antigenically related to the Cl-stimulated cellobiosidase and a proportion of the endoglucanase were released into the extracellular culture fluid during growth, irrespective of the substrate. Immunoelectron microscopy of cells with a polyclonal antibody to Cl-stimulated cellobiosidase as the primary antibody and 10-nm-diameter gold particles conjugated to goat anti-rabbit antibodies as the second antibody revealed protrusions of the outer surface which were selectively labeled with gold, suggesting that Cl-stimulated cellobiosidase was located on the protrusions. These data support the contention that the protrusions have a role in cellulose hydrolysis; however, this interpretation is complicated by reactivity of the antibodies with a large number of other proteins that possess related antigenic epitopes.     

Two beta-glucosidase activities in Fibrobacter succinogenes S85.

Few bacteria are capable of degrading crystalline cellulose but there is considerable interest in the properties of enzyme systems with this capability. In the bovine and ovine rumen the principal cellulolytic bacterium is Fibrobacter (formerly Bacteroides) succinogenes. The cellulase system of this organism is composed of multiple enzyme components, including a constitutive and cell-associated beta-glucosidase active against cellobiose. The properties of the beta-glucosidase activity have been investigated with the chromogenic substrate p-nitrophenyl beta-D-glucoside (pNPG). Hydrolytic activity against pNPG was located primarily in the cytoplasm and the cytoplasmic membrane but showed a gradual migration to the periplasm during growth on either glucose or cellobiose. Activity against cellobiose was found in the periplasm in significant amounts in all growth phases. Of the beta-glucosides tested, only cellobiose and pNPG were hydrolysed by crude cell extracts. In the presence of cellobiose, however, the rate of hydrolysis of pNPG was stimulated up to 10-fold, and extracts hydrolysed methylumbelliferyl beta-D-glucoside, 5-bromo-4-chloro-3-indolyl beta-D-glucoside, arbutin and aesculin. Activities against pNPG in the presence and absence of cellobiose displayed similar instability in the presence of oxygen; both were stabilized by dithiothreitol and the temperature and pH optima were identical. A significant proportion of the membrane-associated beta-glucosidase was released by treatment with 0.3 mol/1 KCl, and fractionation by chromatography on CM-cellulose showed the presence of two activities against pNPG, only one of which was stimulated by cellobiose.     

Isolation and characterization of an endo-beta-galactosidase from Bacteroides fragilis.

Six strains of Bacteroides fragilis were examined and all found to produce endo-beta-galactosidase, an enzyme that hydrolyses internal beta-galactosidic linkages of oligosaccharides belonging to the poly-N-acetyl-lactosamine series, with the common structure GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc/Glc. The enzyme was produced without the addition of an inducer such as keratan sulphate. It was purified 7000-fold from the culture supernatant and obtained with a yield 4-10-fold greater than from sources described previously. The specificity of the enzyme towards bovine corneal keratan sulphate, milk oligosaccharides and the glycolipids lacto-N-neotetraosylceramide and lacto-N-tetraosylceramide closely resembled that of the endo-beta-galactosidase isolated from Escherichia freundii. A novel observation was that both enzymes hydrolysed the type 2 sequence, Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc, at about twice the rate of the type 1 isomer, Gal beta 1 leads to 3GlcNAc beta 1 leads to 3Gal beta 1 leads to 4Glc. Because of the ease of purification of the enzyme and high yield in the absence of contaminating glycosidases and proteinases, Bacteroides fragilis is a valuable source of endo-beta-galactosidase for the structural analysis of carbohydrate chains.     

Catalytic properties of the cellulose-binding endoglucanase F from Fibrobacter succinogenes S85.

The celF gene from the predominant cellulolytic ruminal bacterium Fibrobacter succinogenes encodes a 118.3-kDa cellulose-binding endoglucanase, endoglucanase F (EGF). This enzyme possesses an N-terminal cellulose-binding domain and a C-terminal catalytic domain. The purified catalytic domain displayed an activity profile typical of an endoglucanase, with high catalytic activity on carboxymethyl cellulose and barley beta-glucan. Immunoblotting of EGF and the formerly characterized endoglucanase 2 (EG2) from F. succinogenes with antibodies prepared against each of the enzymes demonstrated that EGF and EG2 contain cross-reactive epitopes. This data in conjunction with evidence that the proteins are the same size, share a 19-residue internal amino acid sequence, possess similar catalytic properties, and both bind to cellulose allows the conclusion that celF codes for EG2.     

Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose

Fibrobacter succinogenes subsp. succinogenes S85, formerly Bacteroides succinogenes, adheres to crystalline cellulose present in the culture medium. When the cells are suspended in buffer, adhesion is enhanced by increasing the ionic strength. Heat, glutaraldehyde, trypsin, and pronase treatments markedly reduce the extent of adhesion. Treatment with dextrinase, modification of amino and carboxyl groups with Formalin or other chemical agents, and inclusion of either albumin (1%) or Tween 80 (0.5%) do not decrease the degree of adhesion. Adherence-defective mutants isolated by their inability to bind to cellulose exhibited different growth characteristics. Class 1 mutants grew on glucose, cellobiose, amorphous cellulose, and crystalline cellulose. Class 3 mutants grew on glucose and cellobiose but not on amorphous or crystalline cellulose. No substantial changes were detected in the endoglucanase, cellobiosidase, and cellobiase activities of the wild type and the mutants. These data suggest that adhesion to crystalline cellulose is specific and that it involves surface proteins.     

Factors affecting adhesion of Fibrobacter succinogenes subsp. succinogenes S85 and adherence-defective mutants to cellulose.

Fibrobacter succinogenes subsp. succinogenes S85, formerly Bacteroides succinogenes, adheres to crystalline cellulose present in the culture medium. When the cells are suspended in buffer, adhesion is enhanced by increasing the ionic strength. Heat, glutaraldehyde, trypsin, and pronase treatments markedly reduce the extent of adhesion. Treatment with dextrinase, modification of amino and carboxyl groups with Formalin or other chemical agents, and inclusion of either albumin (1%) or Tween 80 (0.5%) do not decrease the degree of adhesion. Adherence-defective mutants isolated by their inability to bind to cellulose exhibited different growth characteristics. Class 1 mutants grew on glucose, cellobiose, amorphous cellulose, and crystalline cellulose. Class 3 mutants grew on glucose and cellobiose but not on amorphous or crystalline cellulose. No substantial changes were detected in the endoglucanase, cellobiosidase, and cellobiase activities of the wild type and the mutants. These data suggest that adhesion to crystalline cellulose is specific and that it involves surface proteins.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.