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PubMed1985

Isolation and properties of beta-glucosidase from Ruminococcus albus.

An enzyme active against p-nitrophenyl-beta-D-glucoside was purified from logarithmic-phase cells of Ruminococcus albus cultivated in a medium containing ball-milled cellulose. The purification yielded homogeneous enzyme after an approximately 520-fold increase in specific activity and a 9% yield. The enzyme was identified as a beta-glucosidase because it can hydrolyze cellobiose and cellooligosaccharides to glucose from the nonreducing ends.

Cations, Divalent↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Cloning and expression in Escherichia coli of a cellulase gene from Ruminococcus flavefaciens.

An endoglucanase gene of Ruminococcus flavefaciens FD1 was cloned on the vector pEcoR251 to form the recombinant plasmid pMEB200. The cloned endoglucanase gene showed carboxymethylcellulase enzyme activity but no degradation of Avicel (FMC Corp., Philadelphia, Pa.) or filter paper. Carboxymethylcellulase activity was found during the late-exponential-growth phase and accumulated in the periplasmic fraction. Enzyme production was not subject to catabolite repression by glucose.

Animals↗
PubMed1989

Structure of a Ruminococcus albus endo-1,4-beta-glucanase gene.

A chromosomal DNA fragment encoding an endo-1,4-beta-glucanase I (Eg I) gene from Ruminococcus albus cloned and expressed in Escherichia coli with pUC18 was fully sequenced by the dideoxy-chain termination method. The sequence contained a consensus promoter sequence and a structural amino acid sequence. The initial 43 amino acids of the protein were deduced to be a signal sequence, since they are missing in the mature protein (Eg I). High homology was found when the amino acid sequence of the Eg I was compared with that of endoglucanase E from Clostridium thermocellum. Codon usage of the gene was not biased. These results suggested that the properties of the Eg I gene from R. albus was specified from the known beta-glucanase genes of the other organisms.

Amino Acid Sequence↗
PubMed1991

Modification of the properties of a Ruminococcus albus endo-1,4-beta-glucanase by gene truncation.

An endo-1,4-beta-glucanase (EgI) gene isolated from Ruminococcus albus was deleted at the 5'-flanking region by gene truncation or at the 3'-flanking region by insertion of an omega (omega) fragment with a universal stop codon at the EcoRI or BamHI site. These modified genes were integrated into pUC vectors to construct chimera plasmids for Escherichia coli. The truncated EgIs were produced from transformants (E. coli) harboring the chimera plasmids. An EgI with a 15-amino-acid N-terminal deletion exibited higher activity at lower pH and temperature compared with the activity of the original EgI. The EgIs with 59- and 75-amino-acid deletions from the N and C terminals, respectively, had no activity, indicating that both terminal moieties are essential for enzyme activity.

Amino Acid Sequence↗
PubMed1995

Nucleotide sequence and transcriptional analysis of the celD beta-glucanase gene from Ruminococcus flavefaciens FD-1.

The nucleotide sequence of the celD gene, which encodes endoglucanase and xylanase activity, from Ruminococcus flavefaciens FD-1 was determined. The DNA sequence of celD contains an open reading frame of 1215 nucleotides that encodes a polypeptide of 405 amino acids with a molecular mass of 44,631 Da. The primary amino acid sequence of CelD was screened against the GenBank data base for similar polypeptide sequences and the analysis indicated that CelD has common features with endoglucanases from the family E cellulases. Both hydrophobic cluster and BESTFIT (Genetics Computer Group (University of Wisconsin) package) analyses confirmed this relationship. Pairwise alignments using BESTFIT revealed that CelD was most closely related to endE4 from Thermomonospora fusca over a 160 amino acid window. The histidine, aspartate, and glutamate residues identified as being essential for catalytic activity in family E cellulases are conserved in CelD. A Shine-Dalgarno-like sequence was present 5 base pairs (bp) upstream of the translation start site. Primer extension analysis indicated that different transcription initiation sites are used to initiate transcription of celD in Escherichia coli and R. flavefaciens. In the case of R. flavefaciens the transcription initiation site is at a T residue (nucleotide 273) 16 bp upstream from the translational start site. A region resembling a sigma 70-like-10 promoter sequence is present upstream from the transcription initiation site but there is no apparent-35 region. In contrast, transcription in E. coli is initiated at a C residue 258 bp upstream from the translational start site and a sequence resembling a omega 70-like-10 region is present 5 bp upstream of this residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

Enzymes involved in the formation of 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid from dehydrocholic acid by Ruminococcus sp. obtained from human intestine.

Ruminococcus sp. PO1-3 from human intestinal flora reduced dehydrocholic acid to 3 beta-hydroxy-7,12-dioxo-5 beta-cholanic acid by means of the enzyme 3 beta-hydroxysteroid dehydrogenase (Akao, T., Akao, T., Hattori, M., Namba, T. and Kobashi, K. (1986) J. Biochem. (Tokyo) 99, 1425-1431). This bacterium and its crude extract gave rise to another product, showing a lower RF value on TLC, from dehydrocholic acid. The product was identified as 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid. The crude extract reduced 7-ketolithocholic acid and its methyl ester, but not 6-ketolithocholic acid and 12-ketochenodeoxycholic acid, in the presence of NADPH, and oxidized ursodeoxycholic acid and beta-muricholic acid, but not cholic acid, chenodeoxycholic acid, deoxycholic acid and hydrocholic acid, in the presence of NADP+. Therefore, besides 3 beta-hydroxysteroid dehydrogenase, 7 beta-hydroxysteroid dehydrogenase was shown to be present in this bacterium. The two dehydrogenases were clearly separated from each other by butyl-Toyopearl 650 M column chromatography. From dehydrocholic acid, 7 beta-hydroxy-3,12-dioxo-5 beta-cholanic acid was produced by 7 beta-hydroxysteroid dehydrogenase and 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid was produced by combination of two enzymes, 7 beta- and 3 beta-hydroxysteroid dehydrogenase.

3-Hydroxysteroid Dehydrogenases↗
PubMed1991

Cloning of a beta-glucosidase gene from Ruminococcus albus and its expression in Escherichia coli.

A HindIII fragment of R. albus DNA encoding beta-glucosidase was cloned into E. coli. The DNA sequence (3158 bp) was determined, and the longest potential encoding sequence consisted of 2,841 bp (947 amino acids with the calculated molecular weight of 104,276. The deduced NH2-terminal amino acid sequence from the first (methionine) to the twentieth (glycine) was identical to that of the purified enzyme, suggesting that the gene for beta-glucosidase does not encode a signal peptide. The enzyme purified from the culture supernatant of the transformant had a molecular weight of 120,000 and its maximum activity was revealed at pH 6.5 and 30 degrees C. Reducing reagents activated the enzyme, whereas the sulfhydryl group-blocking reagents and reaction products (glucose) inhibited the activity. Hydrolyzates of celloorigomers contained glucose as a major product, indicating that the enzyme acts as beta-glucosidase. The enzyme from the transformant revealed similar properties to that from R. albus, and both enzyme proteins were immunologically the same to each other, indicating that the cloned gene encodes beta-glucosidase from R. albus.

Amino Acid Sequence↗