Hiroki Ohara

Characterization of the Cellulolytic Complex (Cellulosome) from Ruminococcus albus

The cellulolytic complex was isolated from the culture supernatant of Ruminococcus albus strain F-40 grown on cellulose by a Sephacryl S-300HR column chromatography. The molecular mass of the cellulolytic complex was found to be larger than 1.5×106 Da. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the cellulolytic complex contained at least 15 proteins with molecular weights from 40 kDa to 250 kDa. Among them, 11 proteins showed endoglucanase and/or xylanase activities on the zymograms. Immunological analysis using an antiserum raised against the dockerin domain of endoglucanase VII of R. albus (DocVII) suggested that at least 7 proteins in the cellulolytic complex contained a dockerin domain immunoreactive with the anti-DocVII antiserum. Furthermore, DocVII was shown to specifically interact with a 40-kDa protein of the cellulolytic complex by Far-Western blot analysis. These results strongly suggest that the cellulolytic complex produced by R. albus resembles the cellulosome specified for the cellulolytic complex of several clostridia such as Clostridium thermocellum and respective components are assembled into the cellulosome by the mechanism common in all of the cellulolytic clostridia, i.e., the cellulosome is formed by the interaction between a dockerin domain of catalytic components and a cohesin domain of a scaffolding protein.

Effects of Pro-Hyp, a Collagen Hydrolysate-Derived Peptide, on Hyaluronic Acid Synthesis Using in Vitro Cultured Synovium Cells and Oral Ingestion of Collagen Hydrolysates in a Guinea Pig Model of Osteoarthritis

Proline-hydroxyproline (Pro-Hyp) stimulated hyaluronic acid production in cultured synovium cells. It was detected in guinea pig blood after oral ingestion of collagen hydrolysates. Oral administration of collagen hydrolysates increased the amount of proteoglycans in the epiphyses. It also reduced the morphological changes associated with osteoarthritic cartilage destruction of the knee joint. The results suggest that collagen hydrolysates have therapeutic potential for treatment of osteoarthritis.

Sequence of egV and Properties of EgV, a Ruminococcus albus Endoglucanase Containing a Dockerin Domain

The Ruminococcus albus F-40 egV gene, encoding endoglucanase V (EGV), consists of an open reading frame of 1,833 nucleotides and encodes 611 amino acids with a deduced molecular weight of 67,103. The deduced EGV is a modular enzyme composed of a catalytic domain of family 5 of glycosyl hydrolases, a domain of unknown function, and a dockerin domain responsible for cellulosome assembly, suggesting that R. albus F-40 produces a cellulosome, and EGV is a component of the cellulosome. A truncated form of EGV with an apparent molecular weight of 42,000 was purified from a recounbinant Escherichia coli and characterized since EGV suffered from partial proteolysis by E. coli protease(s). The truncated EGV was active toward carboxylmethyl cellulose, xylan, lichenan, and acid-swollen cellulose. The pH and temperature optima of the enzyme were 7.0 and 40°C, respectively. By Western blot analysis using the antiserum raised against the truncated enzylne, EGV was detected in the whole cells but not in the culture supernatant of R. alubus F-40, suggesting that EGV was located on the cell surface.

Increase in Terminal Restriction Fragments of Bacteroidetes-Derived 16S rRNA Genes after Administration of Short-Chain Fructooligosaccharides

ABSTRACT It is well known that short chain fructooligosaccharides (scFOS) modify intestinal microbiota in animals as well as in humans. Since most murine intestinal bacteria are still uncultured, it is difficult for a culturing method to detect changes in intestinal microbiota after scFOS administration in a mouse model. In this study, we sought markers of positive change in murine intestinal microbiota after scFOS administration using terminal restriction fragment length polymorphism (T-RFLP) analysis, which is a culture-independent method. The T-RFLP profiles showed that six terminal restriction fragments (T-RFs) were significantly increased after scFOS administration. Phylogenetic analysis of the 16S rRNA partial gene sequences of murine fecal bacteria suggested that four of six T-RFs that increased after scFOS administration were derived from the 16S rRNA genes of the class Bacteroidetes. Preliminary quantification of Bacteroidetes by real-time PCR suggests that the 16S rRNA genes derived from Bacteroidetes were increased by scFOS administration. Therefore, the T-RFs derived from Bacteroidetes are good markers of change of murine intestinal microbiota after scFOS administration.

Collagen hydrolysate intake improves the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice.

Ultraviolet B (UVB) irradiation induces serious damage to the skin. Collagen hydrolysate and collagen‐derived peptides have effects on skin function in vivo and in vitro. However, few studies have investigated changes in the epidermal barrier or dermal elasticity caused by UVB. Here, we investigated the loss of epidermal barrier function and skin elasticity induced by UVB irradiation in hairless mice fed collagen hydrolysate.

Isothermal Titration Calorimetric Studies on the Binding of a Family 6 Carbohydrate-binding Module of Clostridium thermocellum XynA with Xlylooligosaccharides

The family 6 carbohydrate-binding module (CBM) of Clostridium thermocellum XynA was expressed, and the binding equilibria of the CBM with xylooligosaccharides (degree of polymerization DP=2−8) were observed by isothermal titration calorimetry (ITC) at pH 8. The association constant, K a, increased with increasing DP from 5×103 M−1 (DP=2) to approximately 5×105 M−1 (DP=5-8) at 20°C. The K a values at 60°C were about 1/10 of those at 20°C. The binding was found to be an enthalpy-driven reaction. The DP dependence of the thermodynamic parameters of the binding reaction suggested the size of the ligand-binding site to be 5 xylose units long.