Keisuke Tanaka

Grimontia hollisae collagenase (Ghcol) exhibits high collagen‐degrading activity. To explore its catalytic mechanism, its substrate (Gly‐Pro‐Hyp‐Gly‐Pro‐Hyp, GPOGPO)‐complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active‐site zinc ion. Since this water was not observed in the product (GPO)‐complexed Ghcol, it was hypothesized that the GPOGPO‐complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active‐site geometry and site‐directed mutagenesis of the active‐site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

Collagenase products are crucial to isolate primary cells in basic research and clinical therapies, where their stability in collagenolytic activity is required. However, currently standard collagenase products fromClostridium histolyticumlack such stability. Previously, we produced a recombinant 74-kDa collagenase fromGrimontia hollisae, which spontaneously became truncated to~60 kDa and possessed no stability. In this study, to generateG. hollisaecollagenase useful as a collagenase product, we designed recombinant 62-kDa collagenase consisting only of the catalytic domain, which exhibits high production efficiency. We demonstrated that this recombinant collagenase is stable and active under physiological conditions. Moreover, it possesses higher specific activity against collagen and cleaves a wider variety of collagens than a standard collagenase product fromC. histolyticum. Furthermore, it dissociated murine pancreata by digesting the collagens within the pancreata in a dose-dependent manner, and this dissociation facilitated isolation of pancreatic islets with masses and numbers comparable to those isolated using the standard collagenase fromC. histolyticum. Implantation of these isolated islets into five diabetic mice led to normalisation of the blood glucose concentrations of all the recipients. These findings suggest that recombinant 62-kDa collagenase fromG. hollisaecan be used as a collagenase product to isolate primary cells.

Flow patterns of capillary entry flow of viscoelastic wormlike micellar fluids are investigated experimentally using a PIV (Particle Image Velocimetry). Two types of velocity distributions are observed for the flow into the capillary depending on the viscoelastic Mach numbers which are defined as the ratio of the average velocity of the capillary flow to the shear wave propagation speed in the fluid. In the case of viscoelastic Mach number less than 1, the flow is almost steady, and the velocity in the core region of the entry flow decreases inversely proportional to the power of the distance from the capillary inlet. While in the case of viscoelastic Mach number greater than 1, the velocity decreases exponentially with the distance. Besides, reversal flows are observed at the supercritical viscoelastic Mach numbers. It is found that the reversal flows develop intermittently alongside the flow into the capillary, causing unsteady flow in the entry region.

The collagenase secreted by Grimontia hollisae strain 1706B is a 74 kDa protein that consists of two parts: the catalytic module and a C‐terminal segment that includes the bacterial pre‐peptidase C‐terminal domain. Here, we produced a recombinant C‐terminal segment protein and examined its ability to bind collagen and other characteristics as compared with collagen‐binding domains (CBDs) derived from Hathewaya histolytica (Clostridium histolyticum) collagenases; these CBDs are the only ones thus far identified in bacterial collagenases. We found that the C‐terminal segment binds to collagen only when the collagen is in its triple‐helical conformation. Moreover, the C‐terminal segment and the CBDs from H. histolytica have comparable characteristics, including binding affinity to type I collagen, substrate spectrum, and binding conditions with respect to salt concentration and pH. However, the C‐terminal segment has a completely different primary structure from those of the CBDs from H. histolytica. As regards secondary structure, in silico prediction indicates that the C‐terminal segment may be homologous to those in CBDs from H. histolytica. Furthermore, we performed collagenase assays using fluorescein isothiocyanate‐labeled type I collagen to show that the C‐terminal segment positively contributes to the collagenolytic activity of the 74 kDa collagenase from G. hollisae.

Collagen is frequently digested using pepsin in industries to produce a triple helical collagen without the N‐ and C‐terminal telopeptides. However, kinetic analysis of this reaction is difficult because several Lys residues in the N‐ and in the C‐terminal telopeptides form covalent bonds, leading to multiple substrates species, and pepsin cleaves collagen at various sites in the N‐terminal and in the C‐terminal telopeptides, yielding different products. Here we performed kinetic analysis of the digestion of bovine type I collagen with porcine pepsin. The reaction could be monitored by SDS‐PAGE by measuring the intensity of the protein bands corresponding to the variant β11 chain. We obtained kinetic parameters relative to the decrease in the variant β11 chain upon digestion. At pH 4.0, the K _m and k _cat values increased with increasing temperature (30 to 65 °C), although the k _cat / K _m values were stable. Additional cleavage at the helical region was detected at 45 to 65 °C. At 37 °C, the K _m and k _cat values increased with decreasing pH, and the k _cat / K _m values at pH 2.1 to 4.5 were stable and higher than those at pH 5.0 and 5.5. No additional cleavage was detected at the examined pH. Thus, the optimal pH and temperatures for selective digestion of collagen telopeptides with pepsin are 2.1 to 4.5 and 30 to 40 °C, respectively. These results suggest that the method might be useful for the kinetic analysis of the digestion of collagen telopeptides with pepsin.

The influence of fiber orientation on the crack propagation behavior was studied with single edgenotched specimens which were cut from an injection-molded plate of short-fiber reinforced plastics of polyphenylenesulphide (PPS) reinforced with 30wt% carbon fibers. Specimens were cut at five fiber angles relative to the molding direction, i.e. ??= 0° (MD), 22.5°, 45°, 67.5°, 90° (TD). Fracture mechanics parameters derived based on anisotropic elasticity were used as a crack driving force. Macroscopic crack propagation path was nearly perpendicular to the loading axis for the cases of MD and TD. For the other fiber angles, the crack path was inclined because the crack tended to propagate along inclined fibers. For mode I crack propagation in MD and TD, the resistance to crack propagation is improved by fiber reinforcement, when the rate is correlated to the range of stress intensity factor. The crack propagation rate, da/dN, was slowest for MD and fastest for TD. For each material, the crack propagation rate is higher for larger R ratio. The effect of R ratio on da/dN diminished in the relation between da/dN and the range of energy release rate, ?GI. Difference among MD, TD and matrix resin becomes small when da/dN correlated to a parameter corresponding the crack-tip radius, H?GI, where H is compliance parameter. Fatigue cracks propagated under mixed loading of mode I and II for the fiber angles other than 0° and 90°. The data of the crack propagation rate correlated to the range of total energy release rate, ?Gtotal, lie between the relations obtained for MD and TD. All data of crack propagation tend to merge a single relation when the rate is correlated to the range of total energy release rate divided by Young’s modulus.