Accelerated degradation of cellulose in silkworm excrement by the interaction of housefly larvae and cellulose-degrading bacteria.

The environmental pollution caused by silkworm (Bombyx mori) excrement is prominent, and rich in refractory cellulose is the bottleneck restricting the efficient recycling of silkworm excrement. This study was performed to investigate the effects of housefly larvae vermicomposting on the biodegradation of cellulose in silkworm excrement. After six days, a 58.90% reduction of cellulose content in treatment groups was observed, which was significantly higher than 11.5% of the control groups without housefly larvae. Three cellulose-degrading bacterial strains were isolated from silkworm excrement, which were identified as Bacillus licheniformis, Bacillus amyloliquefaciens, and Bacillus subtilis based on 16S rRNA gene sequence analysis. These three bacterial stains had a high cellulose degradation index (HC value ranged to between 1.86 and 5.97 and FPase ranged from 5.07 U/mL to 7.31 U/mL). It was found that housefly larvae increased the abundance of cellulose-degrading bacterial genus (Bacillus and Pseudomonas) by regulating the external environmental conditions (temperature and pH). Carbohydrate metabolism was the bacterial communities' primary function during vermicomposting based on the PICRUSt. The results of Tax4Fun indicated that the abundance of endo-ß-1,4-glucanase and exo-ß-1,4-glucanase increased rapidly and maintained at a higher level in silkworm excrement due to the addition of housefly larvae, which contributed to the accelerated degradation of cellulose in silkworm excrement. The finding of this investigation showed that housefly larvae can significantly accelerate the degradation of cellulose in silkworm excrement by increasing the abundance of cellulose-degrading bacterial genera and cellulase.

Identification of a unique 1,4-ß-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-ß-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl ß-D-cellobioside (ρ-NPC) and ρ-nitrophenyl ß-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth. KEY POINTS: • CHU_0961 and its homologues formed a novel group (group C) of GH9 enzymes. • CHU_0961 was identified as a 1,4-ß-d-glucan glucohydrolase with unique properties. • CHU_0961 may play an important role in the early stage of C. hutchinsonii growth.

Production of cellulolytic enzymes by Pleurotus species on lignocellulosic wastes using novel pretreatments.

In the present investigation three species of Pleurotus i.e. P. sajor—caju (P1), P. florida (P2) and P. flabellatus (P3) along with two lignocellulosic substrates namely paddy straw and wheat straw were selected for evaluation of production of extracellular cellulolytic enzymes. During the cultivation of three species of Pleurotus under in vivo condition, the two lignocellulosic substrates were treated with plants extracts (aqueous extracts of ashoka leaves (A) and neem oil (B)), hot water (H) and chemicals (C).Among all treatments, neem oil treated substrates supported better enzyme production followed by aqueous extract of ashoka leaves, hot water and chemical treatment. Between the two substrates paddy straw supported better enzyme production than wheat straw. P. flabellatus showed maximum activity of exoglucanase, endoglucanase and β—glucosidase followed by P. florida and P. sajor—caju.

Functional analysis of the cellulose gene of the pine wood nematode, Bursaphelenchus xylophilus, using RNA interference.

Cellulases are pathogenic substances suspected to be responsible for the development of the early symptoms of nematode disease. The pine wood nematode, Bursaphelenchus xylophilus (Parasitaphelenchidae), is the causal agent of pine wilt disease, which kills millions of pine trees. We used RNA interference (RNAi), a reverse genetic tool, to analyze the function of the endo-ß-1,4-glucanase gene of B. xylophilus, which causes the most serious forest tree disease in China and the rest of eastern Asia. Silencing of this gene was detected through real-time PCR and cellulase activity assays after soaking for 24 h in dsRNA. The cellulase gene silencing effects differed among various siRNAs. The propagation and dispersal ability of these nematodes decreased when the endo-ß-1,4-glucanase gene was silenced. It is important to select an effective siRNA before performing an RNAi test.

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization.

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10-20% SW, 70-80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6-21.8% lignin, 33.1-55.2% cellulose and 14-53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.

Characterization and action patterns of two beta-1,4-glucanases purified from cellulomonas uda CS1-1.

Two beta-1,4-glucanases (DI and DIII fractions) were purified to homogeneity from the culture filtrate of a cellulolytic bacteria, Cellulomonas sp. CS1-1, which was classified as a novel species belonging to Cellulomonas uda based on chemotaxanomic and phylogenetic analyses. The molecular mass was estimated as 50,000 Da and 52,000 Da for DI and DIII, respectively. Moreover, DIII was identified as a glycoprotein with a pI of 3.8, and DI was identified as a non-glycoprotein with a pI of 5.3. When comparing the ratio of the CMC-saccharifying activity and CMC-liquefying activity, DI exhibited a steep slope, characteristic of an endoglucanase, whereas DIII exhibited a low slope, characteristic of an exoglucanase. The substrate specificity of the purified enzymes revealed that DI efficiently hydrolyzed CMC as well as xylan, whereas DIII exhibited a high activity on microcrystalline celluloses, such as Sigmacells. A comparison of the hydrolysis patterns for pNP-glucosides (DP 2-5) using an HPLC analysis demonstrated that the halosidic bond 3 from the nonreducing end was the preferential cleavage site for DI, whereas bond 2, from which the cellobiose unit is split off, was the preferential cleavage site for DIII. The partial N-terminal amino acid sequences for the purified enzymes were 1Ala-Gly-Ser-Thr-Leu-Gln-Ala-Ala-Ala-Ser-Glu-Ser-Gly-Arg-Tyr15- for DI and 1Ala-Asp-Ser-Asp-Phe-Asn-Leu-Tyr-Val-Ala-Glu-Asn-Ala-Met-Lys15- for DIII. The apparent sequences exhibited high sequence similarities with other bacterial beta-1,4-glucanases as well as beta-1,4-xylanases.

Enzymatic Characteristics of a Highly Thermostable ß-(1-4)-Glucanase from Fervidobacterium islandicum AW-1 (KCTC 4680).

A highly thermostable ß-(1-4)-glucanase (NA23_08975) gene (fig) from Fervidobacterium islandicum AW-1, a native-feather degrading thermophilic eubacterium, was cloned and expressed in Escherichia coli. The recombinant FiG (rFiG) protein showed strong activity toward ß-D-glucan from barley (367.0 IU/mg), galactomannan (174.0 IU/mg), and 4-nitrophenyl-cellobioside (66.1 IU/mg), but relatively weak activity was observed with hydroxyethyl cellulose (5.3 IU/mg), carboxymethyl cellulose (2.4 IU/mg), and xylan from oat spelt (1.4 IU/mg). rFiG exhibited optimal activity at 90°C and pH 5.0. In addition, this enzyme was extremely thermostable, showing a half-life of 113 h at 85°C. These results indicate that rFiG could be used for hydrolysis of cellulosic and hemicellulosic biomass substrates for biofuel production.

The family 22 carbohydrate-binding module of bifunctional xylanase/ß-glucanase Xyn10E from Paenibacillus curdlanolyticus B-6 has an important role in lignocellulose degradation.

A newly isolated endo-ß-1,4-xylanase (Xyn10E) from Paenibacillus curdlanolyticus B-6 has a modular structure consisting of a family 22 carbohydrate-binding module (CBM), a glycoside hydrolase (GH) family 10 catalytic domain, two fibronectin type III (Fn3) domains, and a family 3 CBM at the C-terminus. Intact Xyn10E (rXyn10E), CBM22-deleted Xyn10E (X-CBM3), CBM3-deleted Xyn10E (X-CBM22), and GH10 catalytic domain only (X-GH10) were expressed in Escherichia coli. rXyn10E showed bifunctional degradation activity toward xylan and ß-glucan and also degraded microcrystalline cellulose. Although X-CBM3 and X-GH10 had drastically reduced xylanase and ß-glucanase activities, X-CBM22 mostly retained these activities. Similar Km values were obtained for rXyn10E and X-CBM3, but kcat and kcat/Km values for X-CBM3 and X-GH10 were lower than those for rXyn10E, suggesting that CBM22 of Xyn10E may contribute to catalytic efficiency. In binding assays, X-CBM3 was still able to bind to ß-glucan, soluble xylan, insoluble xylan, and cellulose through GH10 and CBM3. These results indicate that CBM22 has an important role not only in binding to xylan and ß-glucan but also in feeding both polysaccharides into the neighboring GH10 catalytic domain. rXyn10E showed remarkable synergism with rXyn11A, a major xylanase subunit of P. curdlanolyticus B-6, in the degradation of untreated corn stover and sugarcane bagasse; however, the combination of X-CBM3 and rXyn11A was not synergistic. These results indicate that Xyn10E and Xyn11A act synergistically on lignocellulosic biomass, and CBM22 is essential for efficient degradation of lignocellulosic materials.

Synergistic growth in bacteria depends on substrate complexity.

Both positive and negative interactions among bacteria take place in the environment. We hypothesize that the complexity of the substrate affects the way bacteria interact with greater cooperation in the presence of recalcitrant substrate. We isolated lignocellulolytic bacteria from salt marsh detritus and compared the growth, metabolic activity and enzyme production of pure cultures to those of three-species mixed cultures in lignocellulose and glucose media. Synergistic growth was common in lignocellulose medium containing carboxyl methyl cellulose, xylan and lignin but absent in glucose medium. Bacterial synergism promoted metabolic activity in synergistic mixed cultures but not the maximal growth rate (µ). Bacterial synergism also promoted the production of ß-1,4-glucosidase but not the production of cellobiohydrolase or ß-1,4-xylosidase. Our results suggest that the chemical complexity of the substrate affects the way bacteria interact. While a complex substrate such as lignocellulose promotes positive interactions and synergistic growth, a labile substrate such as glucose promotes negative interactions and competition. Synergistic interactions among indigenous bacteria are suggested to be important in promoting lignocellulose degradation in the environment.

The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations.

The N-terminal cellulose-binding domains CBDN1 and CBDN2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll beta-sandwich structure with a cleft into which amorphous cellulose and soluble cellooligosaccharides bind. To determine the orientation of the sugar chain within these binding clefts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) spin-labeled derivatives of cellotriose and cellotetraose with isolated CBDN1 and CBDN2 was studied using heteronuclear 1H-15N NMR spectroscopy. Quantitative binding measurements indicate that the TEMPO moiety does not significantly perturb the affinity of the cellooligo-saccharide derivatives for the CBDs. The paramagnetic enhancements of the amide 1HN longitudinal (DeltaR1) and transverse (DeltaR2) relaxation rates were measured by comparing the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hydroxylamine (reduced) forms on the two CBDs. The bound spin-label affects most significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-cellotriose bound to CBDN1. This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBDN1 and CBDN2 in either orientation across their beta-sheet binding clefts. The ratio of the association constants for binding in each of these two orientations is estimated to be within a factor of five to tenfold. This finding is consistent with the approximate symmetry of the hydrogen-bonding groups on both the cellooligosaccharides and the residues forming the binding clefts of the CenC CBDs.

A secretory cellulose-binding protein cDNA cloned from the root-knot nematode (Meloidogyne incognita).

A cDNA encoding a secretory cellulose-binding protein was cloned from the root-knot nematode (Meloidogyne incognita) with RNA fingerprinting. The putative full-length cDNA, named Mi-cpb-1, encoded a 203 amino acid protein containing an N-terminal secretion signal peptide. The C-terminal sequence of the putative MI-CBP-1 was similar to a bacterial-type cellulose-binding domain, whereas the N-terminal sequence did not show significant similarity to any proteins in data bases. Recombinant MI-CBP-1 lacked cellulase activity, but bound to cellulose and plant cell walls. In Southern blot hybridization, Mi-cbp-1 hybridized with genomic DNA from M. incognita, M. arenaria, and M. javanica, but not M. hapla, Heterodera glycines, or Caenorhabditis elegans. Polyclonal antibodies raised against recombinant MI-CBP-1 strongly labeled secretory granules in subventral gland cells of second-stage juveniles in indirect immunofluorescence microscopy. Enzyme-linked immunosorbent assay detection of MI-CBP-1 in stylet secretions of second-stage juveniles with the polyclonal antibodies indicated MI-CBP-1 could be secreted through the nematodes' stylet, suggesting that the cellulose-binding protein may have a role in pathogenesis.

Cloning, sequencing and analysis of the ggh-A gene encoding a 1,4-beta-D-glucan glucohydrolase from Microbispora bispora.

The ggh-A gene, encoding a 1,4-beta-D-glucan glucohydrolase/beta-glucosidase, of Microbispora bispora (Mb) was subcloned and expressed from a 4.0-kb XhoI DNA fragment. The nucleotide sequence of this fragment was determined. Analysis of the sequence revealed one open reading frame (ORF) which encodes a 986-amino-acid (aa) protein with a calculated molecular weight of 107,510. The ggh-A ORF has features typical of an actinomycete gene including high GC content (70.5%) and corresponding biased codon usage. Comparison of the aa sequence of the Mb 1,4-beta-D-glucan glucohydrolase (Mbggh-A) with other glycosidases reveals high overall homology to several beta-glucosidases and a 1,4-beta-D-glucan glucohydrolase belonging to the glycosyl hydrolase family 3. The aa sequence alignments of Mbggh-A and beta-glucosidases show that the active site region potentially involves two Asp residues. The aa sequence homology studies revealed a potential two-domain structure for Mbggh-A and other beta-glucosidases. Furthermore, Mbggh-A has localized homology to a cellulose-binding domain present in some xylanases. This report is significant, as, to date, 1,4-beta-D-glucan glucohydrolases have rarely been reported, though they are assumed to have a critical role in cellulolysis.

A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose.

A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies. Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose. The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase. The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.

[Screening, identifying of cellulose-decomposing strain L-06 and its enzyme-producing conditions].

Cellulases are relatively costly enzymes that are sold in large volumes for use in different industrial applications, and a significant reduction in cost will be important for their commercial use in biorefineries. The production of cellulase is a major factor in the hydrolysis of cellulosic materials. Hence it is essential to make the process economically viable. A strain (L-06) with high cellulase activity was screened from rice straw compost and classified as Penicillium decumbens by the analysis of its morphology and 18S rRNA gene sequences. Different conditions of liquid fermentation medium including nitrogen source, carbon source, surfactant, temperature, initial pH, inoculation quantity for the production of cellulase had been studied. The maximal beta-1, 4-glucosidase(BGL) activity was 1662 u/mL which is 1.49 times of the previous and the maximal exo-beta-1, 4-glucanases(CBH) activity was 2770 u/mL which is 1.36 times of the previous, cultured in the optimal condition for three days. And the maximal endo-beta-1, 4-glucanases (EG) activity was 18064 u/mL which is 1.87 times of the previous and the maximal filter paper enzyme(FPase) activity was 4035 u/mL which is 1.47 times of the previous, cultured in the optimal condition for four days. In the optimization experiments, the EG and CBH in the culture condition (pH10) maintained 70% and 43% activity. In the culture condition (50 degrees C) EG and CBH maintained 59% and 68% activity, which showed heat and alkali resistant characteristics.

Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus--production of extracellular enzymes and characterization of the major cellulases.

Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65% loss of dry mass within 98 days, and it produced endo-1,4-beta-glucanase (EG), endo-1,4-beta-xylanase, endo-1,4-beta-mannanase, 1,4-beta-glucosidase (BG), 1,4-beta-xylosidase, 1,4-beta-mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and beta-glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6-2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed beta-xylosidase and endoxylanase activities. The K(m) for carboxymethylcellulose was 3.5 g l(-1), with the highest activity at pH 3.5 and 70 degrees C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K(m) for p-nitrophenyl-1,4-beta-glucoside was 1.8 mM, with the highest activity at pH 4 and 60 degrees C, and the enzyme was competitively inhibited by glucose (K(i)=5.8 mM). The fungus produced mainly beta-glucosidase and beta-mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and beta-xylosidase were found in fungus-colonized wood.

Purification and properties of a low molecular weight 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp.

A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.

[Comparative role of exo-1,4-beta-glucosidase and cellobiase in the enzymatic hydrolysis of cellulose]. / Sravintel'naia rol' ekzo-1,4-beta-gliukozidazy i tsellobiazy pri fermentativnom gidrolize tselliulozy.

An inhibitory action of glucono-delta-lactone on individual components of cellulase complexes from Trichoderma reesei, T. longibrachiatum, T. lignorum and Aspergillus foetidus has been studied. It was shown that gluconolactone exerts an inhibiting effect on cellobiases only (the inhibition constants varied within the range of 0.03-0.1 mM) and does not influence the activities of endoglucanases, cellobiohydrolases and exoglucosidases of the complexes. This formed a basis for a new method for determination of the exoglucosidase activity in a mixture with other components of the cellulase complexes. The complete and selective inhibition of cellobiases by gluconolactone with exoglucosidases activity being intact allowed to evaluate the relative contribution of these enzymes in glucose formation in the course of enzymatic hydrolysis of cellulose (CM-cellulose, filter paper and Avicel). It was found that for most of the cellulase complexes studied the crucial role in glucose formation both from soluble and insoluble cellulose at early steps of hydrolysis belongs to exoglucosidase. On the other hand, the role of exoglucosidase (comparatively with cellobiase) progressively decreases in the course of cellulose hydrolysis. The latter effect does not presumably reflect the changes in the mechanism of cellulose conversion in the course of hydrolysis, but is due to a specific kinetic behaviour of the multienzyme cellulase system.

Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex.

The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme.

Affinity purification of cellulose-binding enzymes of Clostridium stercorarium.

Cellulose-affinity chromatography proved to be a fast and efficient purification procedure for exoenzymes of the thermophilic anaerobe Clostridium stercorarium, suitable for the preparation of large amounts of enzymes for technical applications. The cellulose-binding enzymes could be identified as the C. stercorarium; cellulolytic enzymes Avicelase I and Avicelase II characterized previously as endo-1,4-beta-glucanase and exo-1,4-beta-glucanase. A third protein was identified as xylanase A, the major endo-1,4-beta-xylanase of this organism.

Purification and characterization of an exo-beta-1,4-glucanase from Ruminococcus flavefaciens FD-1.

An exo-beta-1,4-glucanase (Exo A) from Ruminococcus flavefaciens FD-1 was purified to homogeneity and characterized. Enzyme activity was monitored during purification by using the substrate p-nitrophenyl-beta-D-cellobioside (NPC). Over 85% of the NPC activity was found to be extracellular once the filter paper was degraded (7 days). Culture supernatant was harvested, and the protein was concentrated by ultrafiltration. The retentate (greater than or equal to 300,000 Mr), containing most of the activity against NPC, was then fractionated with a TSK DEAE-5PW column. This yielded a sharp major peak of NPC enzyme activity, followed by a broader, less active area that appeared to contain at least six minor peaks of lower enzymatic activity. Further purification was achieved by chromatography with a hydroxylapatite column. Finally, gel filtration chromatography yielded a homogeneous enzyme (Exo A) as determined by silver stains of both sodium dodecyl sulfate- and nondenaturing electrophoresis gels. Substrate specificity experiments and the products of cellulose digestion indicate that the enzyme was an exo-beta-1,4-glucanase. Exo A required Ca2+ for maximal activity and had an apparent Km of 3.08 mM for NPC, with a Vmax of 0.298 mumol/min per mg of protein. The enzyme had an Mr of 230,000, as determined by gel filtration chromatography, and was a dimer of 118,000-Mr subunits. The N-terminal amino acid sequence of the enzyme is presented.