Clostridium Stercorarium Research Articles

Intramolecular synergism in an engineered exo-endo-1,4-beta-glucanase fusion protein.

Exoglucanase CelY and endoglucanase CelZ from the cellulolytic thermophile Clostridium stercorarium act in synergism to hydrolyse cellulosic substrates. To increase the efficiency of the hydrolytic degradation process, an artificial multienzyme carrying both enzymatic activities on one polypeptide chain was constructed by gene fusion. A segment of CelZ, CelZdeltaBB'C (designated CelZC'), comprising the catalytic domain and the adjacent domain C' homologous to the cellulose-binding domain family IIIc, was fused to the C-terminus of CelY, yielding the fusion protein CelY-CelZC', designated CelYZ. The large fusion protein (170 kDa) could be isolated from a recombinant Escherichia coli strain in its intact form retaining the pronounced thermostability of the fusion partners. As a true multienzmye, CelYZ exhibited both exoglucanase and endoglucanase activities. The cellulolytic activity of the fusion protein was three- to fourfold higher than the sum of the individual activities. Dilution experiments showed that the enhanced cellulolytic activity of the multienzyme resulted from intramolecular synergism of the fusion partners. The product profiles and the kinetic constants of cellulose hydrolysis support a new mechanistic model proposed for explaining the co-operativity of the two catalytic domains within the multienzmye.

Isoprenoid quinone, cellular fatty acid composition and diaminopimelic acid isomers of newly classified thermophilic anaerobic Gram-positive bacteria

The configuration of quinone systems, cellular fatty acids and diaminopimelic acids in 11 species of thermophilic clostridia which have been classified into new genera based on their 16S rRNA sequences was determined. It was found that menaquinone 7 was present in 10 species as the major component of menaquinone systems by analyses using HPLC with photodiode-array detector and electron impact mass spectrometry. In seven of the 11 species, the major cellular fatty acids were determined to be iso-15:0 and iso-17:0. As a results of chemotaxonomic studies it was demonstrated that the representatives of the new genera Thermoanaerobacter and Thermoanaerobacterium were heterogeneous in their chemical composition, whereas strains of the new genus Moorella and of the new unnamed genus which is composed of (Clostridium) stercorarium and (Clostridium) thermolacticum showed homogeneity in their chemotaxonomic characteristics.

Properties and gene structure of a bifunctional cellulolytic enzyme (CelA) from the extreme thermophile ‘Anaerocellum thermophilum’ with separate glycosyl hydrolase family 9 and 48 catalytic domains

A large cellulolytic enzyme (CelA) with the ability to hydrolyse microcrystalline cellulose was isolated from the extremely thermophilic, cellulolytic bacterium 'Anaerocellum thermophilum'. Full-length CelA and a truncated enzyme species designated CelA' were purified to homogeneity from culture supernatants. CelA has an apparent molecular mass of 230 kDa. The enzyme exhibited significant activity towards Avicel and was most active towards soluble substrates such as CM-cellulose (CMC) and beta-glucan. Maximal activity was observed between pH values of 5 and 6 and temperatures of 95 degrees C (CM-cellulase) and 85 degrees C (Avicelase). Cellobiose, glucose and minor amounts of cellotriose were observed as end-products of Avicel degradation. The CelA-encoding gene was isolated from genomic DNA of 'A. thermophilum' by PCR and the nucleotide sequence was determined. The celA gene encodes a protein of 1711 amino acids (190 kDa) starting with the sequence found at the N-terminus of CelA purified from 'A. thermophilum'. Sequence analysis revealed a multidomain structure consisting of two distinct catalytic domains homologous to glycosyl hydrolase families 9 and 48 and three domains homologous to family III cellulose-binding domain linked by Pro-Thr-Ser-rich regions. The enzyme is most closely related to CelA of Caldicellulosiruptor saccharolyticus (sequence identities of 96 and 97% were found for the N- and C-terminal catalytic domains, respectively). Endoglucanase CelZ of Clostridium stercorarium shows 70.4% sequence identity to the N-terminal family 9 domain and exoglucanase CelY from the same organism has 69.2% amino acid identity with the C-terminal family 48 domain. Consistent with this similarity on the primary structure level, the 90 kDa truncated derivative CelA' containing the N-terminal half of CelA exhibited endoglucanase activity and bound to microcrystalline cellulose. Due to the significantly enhanced Avicelase activity of full-length CelA, exoglucanase activity may be ascribed to the C-terminal family 48 catalytic domain.

Adsorption of Clostridium stercorarium xylanase A to insoluble xylan and the importance of the CBDs to xylan hydrolysis

Xylanase A (XynA) from Clostridium stercorarium, which consists of a catalytic domain and two family VI cellulose-binding domains (CBDs) each connected by a linker sequence, was found to bind to insoluble oat-spelt xylan as well as acid-swollen cellulose (ASC). Its adsorption to xylan was greatly dependent on the concentration of the phosphate buffer in the binding assay mixtures. The adsorption of XynA to insoluble xylan proceeded in accordance with a Langmuir-type isotherm. The Ka and [PX] max values of XynA for oat-spelt were 0.17 l/μmol and 5.0 μmol/g, respectively. Removal of the CBDs from XynA abolished the cellulose- and xylan-binding abilities of this enzyme and reduced the enzyme activity toward insoluble xylan but not soluble xylan. These results clearly indicated that the CBDs of XynA play an important role in the hydrolysis of insoluble xylan. Desorption of XynA from the ASC-XynA complex was effected by soluble saccharide solutions such as barley β-glucan, birchwood xylan, and glucose solutions as well as a cellobiose solution, indicating that the CBDs of XynA have an affinity for soluble saccharides in addition to insoluble polysaccharides.

Cloning, sequencing and expression of the cellobiose phosphorylase gene of Cellvibrio gilvus

The cellobiose phosphorylase gene of Cellvibrio gilvus was cloned and sequenced. The gene had a high GC content of 70.6% and encoded a protein with 822 amino acid residues. It was expressed in Escherichia coli JM 109 to yield the active enzyme. Based on the N-terminal amino acid sequence of the native enzyme, it is found that the enzyme is produced without a signal peptide. Only two previously reported enzymes showed significant amino acid sequence homology; cellobiose phosphorylase of Clostridium stercorarium (61.4%), and cellodextrin phosphorylase also of C. stercorarium (38.2%).

Purification and Properties of a Cellobiose Phosphorylase (CepA) and a Cellodextrin Phosphorylase (CepB) from the Cellulolytic Thermophile Clostridium Stercorarium

Two phosphorolytic enzymes displaying activity towards the soluble cellulose degradation products cellobiose and cellodextrins were purified from the crude extract of the cellulolytic thermophile Clostridium stercorarium. Both phosphorylases have monomeric structures with molecular masses of 93 and 91 kDa, respectively. Although the N-terminal amino acid sequences are highly similar, a clear distinction of the two enzymes could be made on the basis of their substrate specificities: the enzyme designated cellobiose phosphorylase cleaved exclusively the disaccharide substrate, whereas the enzyme designated cellodextrin phosphorylase accepted only oligosaccharides as substrates. Kinetic constants were determined for the cleavage of cellobiose and cellodextrins. Maximal activity was observed at 65 degrees C in the pH range 6.0-7.0 for both enzymes. The sequences of the genes cepA and cepB encoding the cellobiose phosphorylase and the cellodextrin phosphorylase, respectively, have been submitted to the GenBank database.

Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-beta-glucanase Avicelase II.

The nucleotide sequence of the celY gene coding for the thermostable exo-1,4-beta-glucanase Avicelase II of Clostridium stercorarium was determined. The gene consists of an ORF of 274Z bp which encodes a preprotein of 914 amino acids with a molecular mass of 103 kDa. The signal-peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase II purified from C stercorarium. The celY gene is located in close vicinity to the celZ gene coding for the endo-1,4-beta-glucanase Avicelase I. The CelY-encoding sequence was isolated from genomic DNA of C. stercorarium with the PCR technique. The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion protein could be purified using a simple two-step procedure. The properties of CelY proved to be consistent with those of Avicelase II purified from C. stercorarium. Sequence comparison revealed that CelY consists of an N-terminal catalytic domain flanked by a domain of 95 amino acids with unknown function joined to a type III cellulose-binding domain. The catalytic domain belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).

Synergistic interaction of the Clostridium stercorarium cellulases Avicelase I (CelZ) and Avicelase II (CelY) in the degradation of microcrystalline cellulose

Avicelase I and Avicelase II purified from the cellulolytic thermophile Clostridium stercorarium acted in synergism to hydrolyze microcrystalline cellulose. The degree of synergism proved to be dependent on the ratio of the two enzymes and on the type of the cellulosic substrate. The activity of the combined enzymes towards Avicel was about double the sum of the individual activities. No synergism was found with amorphous cellulose preparations. It is shown that the simultaneous concerted action of both Avicelases is required to observe synergism. We suggest that synergism results from an exo-exo type cooperativity and present a mechanistic model explaining the synergistic interaction between Avicelase I and Avicelase II.

High expression of the xylanase B gene from Clostridium stercorarium in tobacco cells

The xylanase gene ( xynB) from Clostridium stercorarium was expressed in tobacco cells (BY2) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The gene product, xylanase B (XynB), was proteolytically processed in the transformed cells (termed B1-11): a larger form of the protein was mainly found in the culture supernatant and a smaller one was found in the cells. The amount of XynB protein was estimated to be 5% of the total soluble proteins in B1-11 calli after 2 weeks of cultivation. XynB isolated from the transformed tobacco cells had the activity to hydrolyze the cell wall of barley straw.

Purification of the Ruminococcus albus endoglucanase IV using a cellulose-binding domain as an affinity tag

The gene encoding the single cellulose-binding domain II (CBD II) of Clostridium stercorarium xylanase A was fused to the egIV gene encoding endoglucanase IV (EGIV) from Ruminococcus albus. The fusion protein (EGIV + CBDII) expressed in Escherichia coli can be readily purified from the cell-free extract of E. coli in a single step using the affinity of CBD to cellulose. The purified enzyme was cleaved into two moieties, i.e. the catalytic domain and CBD, at a specific site in the linker region by partial digestion with trypsin at 4°C. This result indicates that this CBD belonging to family VI of CBD families can be used as an affinity tag for purification of the recombinant protein.

Characterization of thermostability of Clostridium stercorarium xylanase.

Characterization of thermostability of Clostridium stercorarium xylanase.

Cellulose-binding domains confer an enhanced activity against insoluble cellulose to Ruminococcus albus endoglucanase IV

The gene encoding cellulose-binding domains (CBDs) from Clostridium stercorarium xylanase A was joined to the egIV gene encoding endoglucanase IV (EGIV) from Ruminococcus albus. The hybrid protein (EGIV + CBD) expressed from this fusion gene in Escherichia coli acquired the ability to adsorb onto insoluble celluloses such as ball-milled cellulose (BMC). EGIV + CBD was more active toward BMC at low concentrations than the parental enzyme, EGIV, although there was no difference in the catalytic properties between them toward carboxymethyl cellulose. This result indicates that the addition of the CBDs to EGIV enhances enzyme activity on the insoluble cellulose by concentrating the catalytic domain on the substrate surface.

Debranching of arabinoxylan: properties of the thermoactive recombinant alpha-L-arabinofuranosidase from Clostridium stercorarium (ArfB).

The gene arfB encoding alpha-L-arabinofuranosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment. The recombinant gene product ArfB was purified by fast-performance liquid chromatography. It has a tetrameric structure with a monomeric relative molecular mass of 5200. The optima for temperature and pH are 70 degrees C and 5.0 respectively. The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents. It hydrolyzes aryl and alkyl alpha-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced by recombinant endoxylanase XynA from the same organism. The identify of the N-terminal amino acid sequences indicates that ArfB corresponds to the major alpha-arabinosidase activity present in the culture supernatant of C. stercorarium.

Alpha-D-glucuronidases from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum.

alpha-D-Glucuronidases were purified from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum. This enzyme activity was found to be intracellular in each organism, with T. saccharolyticum producing much greater total activity. The specific activities of the purified enzymes (10 U mg-1 T. saccharolyticum; 1.7 U mg-1 C. stercorarium) differed by a factor of approximately 5. For the determination of enzyme activities, 4-O-methyl-alpha-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by alpha-D-glucuronidase action was quantified by a colorimetric procedure. 4-O-Methyl-alpha-D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of 4-O-methyl-alpha-D-glucuronoxylan with xylanases of C. stercorarium. Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected. Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-nitrophenyl-alpha-D-glucuronoside. Both alpha-D-glucuronidases have a dimeric structure, with monomeric molecular masses of 72 and 76 kDa for C. stercorarium and of 71 kDa for T. saccharolyticum. The pI was estimated to be 4.3 for each enzyme. While both enzymes exhibited a similar pH optimum (pH 5.5-6.5) they differed in their thermostabilities. At 60 degrees C, half-lives of 14 and 2.5 h, respectively, were determined for the alpha-D-glucuronidases of C. stercorarium and T. saccharolyticum. This description of alpha-D-glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi.

A xylan hydrolase gene cluster in Prevotella ruminicola B(1)4: sequence relationships, synergistic interactions, and oxygen sensitivity of a novel enzyme with exoxylanase and beta-(1,4)-xylosidase activities

Two genes concerned with xylan degradation were found to be closely linked in the ruminal anaerobe Prevotella ruminicola B(1)4, being separated by an intergenic region of 75 nucleotides. xynA is shown to encode a family F endoxylanase of 369 amino acids, including a putative amino-terminal signal peptide. xynB encodes an enzyme of 319 amino acids, with no obvious signal peptide, that shows 68% amino acid identity with the xsa product of Bacteroides ovatus and 31% amino acid identity with a beta-xylosidase from Clostridium stercorarium; together, these three enzymes define a new family of beta-(1,4)-glycosidases. The activity of the cloned P. ruminicola xynB gene product, but not that of the xynA gene product, shows considerable sensitivity to oxygen. Studied under anaerobic conditions, the XynB enzyme was found to act as an exoxylanase, releasing xylose from substrates including xylobiose, xylopentaose, and birch wood xylan, but was relatively inactive against oat spelt xylan. A high degree of synergy (up to 10-fold stimulation) was found with respect to the release of reducing sugars from oat spelt xylan when XynB was combined with the XynA endoxylanase from P. ruminicola B(1)4 or with endoxylanases from the cellulolytic rumen anaerobe Ruminococcus flavefaciens 17. Pretreatment with a fungal arabinofuranosidase also stimulated reducing-sugar release from xylans by XynB. In P. ruminicola the XynA and XynB enzymes may act sequentially in the breakdown of xylan.

Process of thermal denaturation of xylanase (XynB) from Clostridium stercorarium F-9.

The thermal denaturation process of Clostridium stercorarium F-9 xylanase (XynB) was studied by monitoring remaining activity and recovered activity of the enzyme. At pH 5.5, aggregation occurred rapidly after the thermal denaturation initiated. The aggregated protein could be dissolved in 8 M urea solution, and the enzyme activity was recovered by diluting the urea. The extent of the recovered activity was gradually decreased with two phases as the reaction time of the thermal denaturation became longer. These results suggested the thermal denaturation process to be as follows: [formula: see text] where N is the native state of the enzyme; D1 is the denatured state of the enzyme that is formed rapidly after the reaction started and can be renatured by the urea treatment, and D2 and D3 are the denatured states of the enzyme that cannot be renatured even by the urea treatment. The rate constants were k1 > 9.2, k2 = 0.33, and k2 = 0.57, and k3 = 0.13 (in min-1 unit).

Molecular Characterization of Four Strains of the Cellulolytic ThermophileClostridium Stercorarium

Four strains of Clostridium stercorarium, including three isolates with increased capacity to hydrolyze crystalline cellulose and assigned to the species, were characterized using recombinant DNA techniques, and compared to C. thermocellum. Their genomic DNA contained 3.0 megabases with 43% G+C. The genomic DNA fragment pattern was identified for the restriction endonucleases NotI and SfiI. Key enzymes of cellulolysis and hemicellulolysis were shown to be present in the genomic DNA of the new strains. A partial physical genomic map containing 6 genes involved in polysaccharide degradation for the type strain and 4 genes for the new isolates is presented. Strains of the species characteristically produced two extracellular cellulose binding proteins representing Avicelase I and II. The methods described allow us to ascribe the newly isolated thermophilic cellulolytic bacteria to the species C. stercorarium and are a basis for molecular biological work on that organism.

Purification and characterization of xylanase A from Clostridium stercorarium F-9 and a recombinant Escherichia coli.

Xylanase A encoded by the Clostridium stercorarium F-9 xynA gene was purified to homogeneity from a recombinant clone of Escherichia coli. The N-terminal amino acid sequence and molecular weight (54,000) estimated by SDS-PAGE of the purified enzyme were consistent with those deduced from the nucleotide sequence [Biosci. Biotech. Biochem., 57, 273-277 (1993)]. A xylanase was also purified to homogeneity from a culture supernatant of C. stercorarium F-9. Its N-terminal amino acid sequence, molecular weight, and enzymatic properties were quite in agreement with those of the recombinant enzyme, indicating that the xynA gene was predominantly expressed as a xylanase gene in C. stercorarium F-9. The purified enzyme hydrolyzed xylotriose to yield xylobiose and xylose while it was less active toward xylobiose. It was optimally active at 75 degrees C and pH 7.0 Km and Vmax were estimated to be 1.9 mg/ml and 2.8 mumol of xylose equivalent/min/micrograms for oat spelt xylan, respectively.

Nucleotide sequence of the Clostridium stercorarium xynA gene encoding xylanase A: identification of catalytic and cellulose binding domains.

The nucleotides of the xynA gene of Clostridium stercorarium were sequenced. The structural gene consists of an open reading frame of 1533 bp encoding 511 amino acids with an M(r) of 56,519. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of the enzyme produced by a recombinant Escherichia coli. Xylanase A consists of a catalytic domain belonging to family G at the N-terminus and two direct repeats of about 90 amino acids with a short spacing at the C-terminus. Deletion analysis showed that the repeated sequences were responsible for binding the enzyme to Avicel and were not essential for catalytic activity. The catalytic domain of this enzyme is highly homologous to xylanase A of Clostridium acetobutylicum (identity: 69%) and xylanase B of Bacillus pumilus (identity: 64%).

Nucleotide Sequence of the Clostridium stercorarium xylA Gene Encoding a Bifunctional Protein with β-D-Xylosidase and α-L-Arabinofuranosidase Activities, and Properties of the Translated Product.

The nucleotides of the β-xylosidase (xylA) gene from Clostridium stercorarium were sequenced. A single open reading frame of 473 codons specifying the subunit (MW 53,340) of xylosidase was identified. The N-terminal amino acid sequence and molecular weight estimated by SDS-polyacrylamide gel electrophoresis of the purified enzyme were quite in agreement with those deduced from the nucleotide sequence. Analysis of the enzyme by gel filtration on an HPLC column gave a molecular weight of 220,000, suggesting that the native enzyme is a tetramer composed of 4 identical subunits. The pH optimum was 7.0 and quite stable over the pH range of 5 to 10 at 4°C. The optimum temperature was 65°C. Vm was estimated to be 5.9nmol/min/μg for p-nitrophenyl-β-D-xylopyranoside and 16.7nmol/min/μg for p-nitrophenyl-α-L-arabinofuranoside, while Km was estimated to be 2.5 mM for p-nitrophenyl-β-D-xylopyranoside and 17.6 mM for p-nitrophenyl-α-L-arabinofuranoside.