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.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

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.     

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.     

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.     

Fibrobacter succinogenes S85 has multiple xylanase genes

Four distinct DNA fragments encoding xylanase activities, pBX1.2, pXC30.2, pX14 and LX31, were cloned from plasmid and γ libraries constructed using genomic DNA from Fibrobacter succinogenes S85. pBX1.2 contained an insert which was homologous, and mapped similarly to that previously cloned in pBX1 while the three remaining clones pX14, pXC30 in plasmids, and LX31 in lambda, represented new xylanase activities. The X14 xylanase was a 73 kDa exo-type xylanase, which was exported to the periplasm of the Escherichia coli host, and produced large quantities of xylose and xylobiose from oat spelt xylan. The XC30 xylanase, also exported in E. coli, was a 77 kDa protein which exhibited both xylanase and endoglucanase activities, and a low cellobiosidase activity. The LX31 enzyme was a 58 kDa endoxylanase that produced a mixture of xylooligosaccharides. Zymograms of isoelectric focusing gels showed that the X14 xylanase had a neutral pI, XC30 contained acidic, neutral and basic enzymic components, while BX1 and LX31 were acidic. These results indicate that, in addition to the many other elements of its polysaccharide-degrading repertoire, F. succinogenes S85 possesses at least four distinct xylanases.     

A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85

We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenes S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25 degrees C, and retains about 70% of its maximum activity at 0 degrees C, while EGD has a temperature optimum of 35 degrees C and retains only about 18% of its maximal activity at 0 degrees C. When assayed at 4 degrees C, CelG exhibits a 33-fold-higher kcat value and a 73-fold-higher physiological efficiency (kcat/Km) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or that F. succinogenes originated from the marine environment.     

Kinetics of Cellulose Digestion by Fibrobacter succinogenes S85

Growing cultures of Fibrobacter succinogenes S85 digested cellulose at a rapid rate, but nongrowing cells and cell extracts did not have detectable crystalline cellulase activity. Cells that had been growing exponentially on cellobiose initiated cellulose digestion and succinate production immediately, and cellulose-dependent succinate production could be used as an index of enzyme activity against crystalline cellulose. Cells incubated with cellulose never produced detectable cellobiose, and cells that were preincubated for a short time with thiocellobiose lost their ability to digest cellulose (competitive inhibition [K(infi)] of only 0.2 mg/ml or 0.56 mM). Based on these results, the crystalline cellulases of F. succinogenes were very sensitive to feedback inhibition. Different cellulose sources bound different amounts of Congo red, and the binding capacity was HCl-regenerated cellulose > ball-milled cellulose > Sigmacel > Avicel > filter paper. Congo red binding capacity was highly correlated with the maximum rates of metabolism of cellulose digestion and inversely related to K(infm). Congo red (250 (mu)g/ml) did not inhibit the growth of F. succinogenes S85 on cellobiose, but this concentration of Congo red inhibited the rate of ball-milled cellulose digestion. A Lineweaver-Burk plot of ball-milled cellulose digestion rate versus the amount of cellulose indicated that Congo red was a competitive inhibitor of cellulose digestion (K(infi) was 250 (mu)g/ml).     

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.     

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.     

Characterization of a family 45 glycosyl hydrolase from Fibrobacter succinogenes S85

Fibrobacter succinogenes is one of the most active cellulolytic bacteria ever isolated from the rumen, but enzymes from F. succinogenes capable of hydrolyzing native (insoluble) cellulose at a rapid rate have not been identified. However, the genome sequence of F. succinogenes is now available, and it was hoped that this information would yield new insights into the mechanism of cellulose digestion. The genome has a single family 45 beta-glucanase gene, and some of the enzymes in this family have good activity against native cellulose. The gene encoding the family 45 glycosyl hydrolase from F. succinogenes S85 was cloned into Escherichia coli JM109(DE3) using pMAL-c2 as a vector. Recombinant E. coli cells produced a soluble fusion protein (MAL-F45) that was purified on a maltose affinity column and characterized. MAL-F45 was most active on carboxymethylcellulose between pH 6 and 7 and it hydrolyzed cellopentaose and cellohexaose but not cellotetraose. It also cleaved p-nitrophenyl-cellopentose into cellotriose and p-nitrophenyl-cellobiose. MAL-F45 produced cellobiose, cellotriose and cellotetraose from acid swollen cellulose and bacterial cellulose, but the rate of this hydrolysis was much too low to explain the rate of cellulose digestion by growing cultures. Because the F. succinogenes S85 genome lacks dockerin and cohesin sequences, does not encode any known processive cellulases, and most of its endoglucanase genes do not encode carbohydrate binding modules, it appears that F. succinogenes has a novel mechanism of cellulose degradation.     

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.     

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.     

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.     

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.     

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.     

Type II DNA restriction-modification system and an endonuclease from the ruminal bacterium Fibrobacter succinogenes S85.

Fibrobacter succinogenes is an important cellulolytic bacterium found in the rumen and cecum of herbivores. Numerous attempts to introduce foreign DNA into F. succinogenes S85 have failed, suggesting the presence of genetic barriers in this organism. Results from this study clearly demonstrate that F. succinogenes S85 possesses a type II restriction endonuclease, FsuI, which recognizes the sequence 5'-GG(A/T)CC-3'. Analysis of the restriction products on sequencing gels showed that FsuI cleaves between the two deoxyguanosine residues, yielding a 3-base 5' protruding end. These data demonstrate that FsuI is an isoschizomer of AvaII. A methyltransferase activity has been identified in the cell extract of F. succinogenes S85. This activity modified DNA in vitro and protected the DNA from the restriction by FsuI and AvaII. DNA modified in vivo by a cloned methylase gene, which codes for M.Eco47II, also protected the DNA from restriction by FsuI, suggesting that FsuI is inhibited by methylation at one or both deoxycytosine residues of the recognition sequence. The methyltransferase activity in F. succinogenes S85 is likely modifying the same deoxycytosine residues, but the exact site(s) is unknown. A highly active DNase (DNase A) was also isolated from the cell extract of this organism. DNase A is an endonuclease which showed high activity on all forms of DNA (single stranded, double-stranded, linear, and circular) but no activity on RNA. In vitro, the DNase A hydrolyzed F. succinogenes S85 DNA extensively, indicating the lack of protection against hydrolysis by this enzyme. In the presence of Mg2+, DNA was hydrolyzed to fragments of 8 to 10 nucleotides in length. The presence of DNase A and the type II restriction-modification system of F. succinogenes S85 may be the barriers preventing the introduction of foreign DNA into this bacterium.     

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.     

Characterization of the NADH dehydrogenase and fumarate reductase of Fibrobacter succinogenes subsp. succinogenes S85

Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K⁺ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K _i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K _i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K _m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201     

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.