Ayako Nishida

Insolubilizing and adhesive studies of water soluble synthetic model proteins

Insolubilizing and adhesive studies of water-soluble synthetic copoly(Tyr1 Lysx) (x = 1-10) were examined using tyrosinase in water and simulated seawater systems. Tyrosinase oxidized tyrosine aromatic nuclei, causing intermolecular crosslinking reactions, which have been assigned by the absorption band at around 360 nm. The viscosities of the model polypeptides were affected by salinity and the kinds of salts in solution systems. As a whole the amino acid compositions, salinity, system pH and beta-structure conformation are considered to play roles in the insolubilizing reaction. The bonding strengths of the model polypeptides exhibited tensile strengths of 16-24 kg/cm2 without enzyme and 29-31 kg/cm2 with tyrosinase on iron, and increased up to 10 kg/cm2 on metals by the addition of tyrosinase as an oxidant.

Preparation of Pure Cellulose Nanofiber via Electrospinning

We describe the preparation of cellulose nanofibrous material directly from cellulose solution via electrospinning. The resulting nanofiber is composed of pure cellulose. The spinning procedure can be performed under ambient conditions at room temperature without post-spun treatment. By mixing drugs with the pre-spun cellulose solution, followed by electrospinning, the drug-loaded nanofiber was prepared and the releasing properties were examined with respect to biomedical applications.

A Glycosylated Byssal Precursor Protein from the Green Mussel Perna viridis with Modified Dopa Side-chains

Foot tissue of the green mussel Perna viridis contains a variety of byssal precursor proteins with the unusual redox-active amino acid, Dopa (L-β-3,4-dihydroxyphenyl-α-alanine). Eight proteins were detectable in acidic extracts of the Perna foot by a redox cycling assay with nitroblue tetrazolium. In one of these, however, P. viridis foot protein-1 (Pvfp-1), activity was not due to Dopa, but to another redox-active derivative. Based on specific colorimetric derivatization with Arnows reagent, ninhydrin and phenylisothiocyanate (Edman), mass spectrometry, the redox-active derivative in Pvfp-1 is not consistent with any known modification. Another uncommon modification of Pvfp-1 involves O-glycosylation of threonine by mannose, glucose or fucose. As in previously characterized fp-1s, the primary sequence of the Pvfp-1 (apparent mass 89 kDa) has two consensus decapeptide motifs; one is APPKPX1TAX2K and the other is APPPAX1TAX2K, where P is Pro/Hyp, and X1 and X2 are difucosylated threonine and a redox sensitive derivative of tyrosine or Dopa, respectively. Of these two unusual residues, X2 is unique to Pvfp-1, whereas O-glycosylated Thr has been previously detected in freshwater mussel fp-1. The sequence homology of Pvfp-1 with the common structural motifs of the fp-1 protein family strongly suggests that the Pvfp-1 functions as the byssal coating (lacquer) protein.

Studies on fouling by the freshwater mussel limnoperna fortunei and the antifouling effects of low energy surfaces

Attachment of the freshwater mussel, Limnoperna fortunei, was tested using non‐treated surfaces, viz. glass, nylon, rubber, silicone and Teflon, together with glass surfaces modified with nine kinds of silane coupling agents. Among the surfaces tested, the mussel avoided attaching to Teflon, silicone, and glass modified with 3‐bromopropyltrimethoxysilane or 3,3,3‐(trifluo‐ropropyl)‐trimethoxysilane. With respect to the relationship between the percentage attachment and the surface free energy (sfe) of the substrates, it was found that attachment was considerably reduced on the substrates which exhibited relatively low sfe, as above. The mean number of secreted byssuses per attaching mussel also decreased with decreasing substrate sfe. Furthermore, when the sfe was divided into the dispersion and polar components, the percentage mussel attachment was related to the polar component. These results suggest that effective antifouling towards L. fortunei is achieved on substrates with a low sfe polar component.

Polyion complex fiber and capsule formed by self-assembly of poly-L-lysine and gellan at solution interfaces

Different characteristic surface structures such as capsules, regularly spaced droplets, and fibers are formed by electrostatic interaction between poly-L-lysine (PLL) and gellan gum via polyion complex (PIC) formation. Spherical droplet PIC capsules of varying diameters form in solutions. Some dyes adsorb on the surface of the capsules, and other dyes penetrate into the capsules. The strong PIC fiber can be spinnable by gravity and by wet spinning in ethanol. This fiber possesses a counterion pairing structure and exhibits the nervation/veining pattern and hollow yarn. The tensile strength of the fiber is 27.8 kg/mm2 [1.40 g/denier (d)] and the knotting strength is 9.98 kg/mm2 (1.13 g/d). By using an organic crosslinking agent, epichlorohydrin, the tensile strength can be increased to 38.5 kg/mm2 (2.46 g/d) and the knotting strength can be increased to 12.2 kg/mm2 (1.99 g/d). The PIC fiber can be dyed by five different dyeing procedures such as direct and vat dyeings. The PLL PIC fiber is water insoluble and has potential as a new synthetic polypeptide fiber technology.

Green mussel Perna viridis L.: attachment behaviour and preparation of antifouling surfaces.

The green mussel Perna viridis LINNE can be kept in simulated seawater for more than 6 months in good condition. The mussel forms many threads by secreting an adhesive protein from the foot, and attaches with more than 50 byssal threads, which makes most mussels clump together. In order to investigate the preparation of the antifouling surfaces toward green mussels, the attachment of mussels was tested using glass surfaces modified with silane coupling agents, together with non-treated material surfaces such as glass and silicone. The correlation between the attachment percentage and the mean number of the secreted byssus was highly significant, indicating that the mussel selects a favorable surface prior to the secretion of byssus. The relationships between the mussel attachment and the surface chemical parameters (surface free energy (sfe) and its dispersion and polar components) were examined based on a working hypothesis, which we have previously reported. The result of statistical regression test indicated that a certain correlation was found between the dispersion component and the mussel attachment, while the polar component did not correlate to the mussel attachment. The present surface chemical approach provided an additional clue for the preparation of ecologically clean antifouling materials that takes into account the combination of the wettability of both the marine adhesive proteins (MAP) and the modified surfaces.

Cross-linking and gel formation of water-soluble lysine polypeptides. An insolubilization model reaction for adhesive proteins

Insolubilizing studies of water-soluble synthetic polypeptides containing lysine residues were examined using organic aliphatic and aromatic cross-linking agents such as dialdehydes, diacyl chlorides and diactive ester, together with an enzyme tyrosinase, in water and simulated seawater systems. The cross-linking reaction was characterized by the viscosity and turbidity changes. Among the organic cross-linking agents used aliphatic glutaraldehyde and aromatic o-phthalaldehyde were the most effective. When excess organic cross-linking agents were added to the lysine polypeptide systems, the corresponding solid gels were formed. As a whole, the molecular weight of the samples, the amino acid compositions, the cross-linking agent used, the molar ratios between cross-linking agents and functional residues and system pH were found to have roles in the insolubilizing reaction and the gel formation. The cross-linking results obtained were compared with those of the polypeptide-tyrosinase systems, whose deep brownish red colour was decolorized by the addition of L-ascorbic acid.

Biodegradation of Chitosan-Gellan and Poly(L-lysine)-Gellan Polyion Complex Fibers by Pure Cultures of Soil Filamentous Fungi

The degradation of two kinds of polyion complex (PIC) fibers, chitosan-gellan (CGF), and poly(L-lysine)-gellan (LGF) fibers, by seven species of soil filamentous fungi has been investigated. All of the pure-line soil filamentous fungi, Aspergillus oryzae, Penicillium caseicolum, P. citrinum, Mucor sp., Rhizopus sp., Curvularia sp., and Cladosporium sp. grew on the two fiber materials. Microscopic observation of the biodegradation processes revealed that P. caseicolum on the CGF and LGF grew, along with the accompanying collapse of the fiber matrices. In the biochemical oxygen-demand (BOD) test, the biodegradation of the LGF by P. caseicolum and Curvularia sp. exceeded 97% carbon dioxide generation and the biodegradation of the CGF by A. oryzae was 59%. These results might offer some clues to the applications of the PIC fibers as environmentally biodegradable materials.

Photoresponsive Peptide and Polypeptide Systems, 14. Biodegradation of Photocrosslinkable Copolypeptide Hydrogels ContainingL-Ornithine andδ-7-Coumaryloxyacetyl-L-ornithine Residues

Copoly[Orn/Orn(Cou)] containing δ-7-coumaryloxyacetyl-L-ornithine [Orn(Cou)] and L-ornithine (Orn) residues was synthesized by the N-carboxyanhydride method. When aqueous solutions of copoly[Orn/ Orn(Cou)] containing 5-10 mol-% of Orn(Cou) are irradiated, the photoinduced dimerization reaction between coumarin moieties in the side chains proceeds slowly, and after 24 h the solutions become transparent hydrogels. The gels exhibit solvent-induced reversible expansion and contraction behavior in both water and ethanol. The biodegradation of the bydrogels by proteolytic enzymes and soil filamentous fungi is investigated using photocrosslinked copoly[Orn/Orn(Cou)] gels. The copoly[Orn 89 / Orn(Cou) 11 ] gel is degradable by protease type XXIII, but not by trypsin. In the biochemical oxygen demand test, the order of the microbial biodegradation (%) was Rhizopus sp. (92%) > A. oryzae (38%) > P. caseicolum (18%) > P. citricum (11%) > Cladosporium sp. (6%). The order for the copoly[Orn 89 /Orn(Cou) 1 ] hydrogel is inverse to that for a polylysine/glutaraldehyde gel. These results suggest that the biodegradabilities of photocrosslinked hydrogels can be controlled by the menomer ratio of Orn, Orn(Cou) and lysine (Lys) in the parent copoly(amino acid)s of the photocrosslinked hydrogels.

Purification of DOPA-Containing Foot Proteins from Green Mussel, Perna viridis, and Adhesive Properties of Synthetic Model Copolypeptides

The thread-like adhesive tissue of the green mussel, Perna viridis, is referred to as the byssus. The phenol gland-derived proteins are involved in the underwater adhesion of the byssus. The objectives of the present study are identification and characterization of the phenol gland-derived proteins in the foot of P. viridis. P. viridis foot contains at least eight kinds of potential precursor proteins, designated as P. viridis foot proteins (Pvfp)-a to -h. Among the precursors, Pvfp-g and Pvfp-h are found at relatively high levels in the phenol gland. These proteins are considered to be essential components in the adhesive plaque, which mediates adhesion of the thread to the substrate surface. Both Pvfp-g and Pvfp-h are enriched in L-β-3,4-dihydroxyphenyl-α-alanine (DOPA), tyrosine (Tyr) and cysteine (Cys), as well as glycine (Gly) and lysine (Lys). According to the amino acid compositions in Pvfp-g and Pvfp-h, two copolypeptides containing four amino acids, Cys, Tyr, Gly, and Lys, were synthesized as the biomimetic model materials. The copolypeptides were subjected to the tyrosinase-catalyzed oxidation, followed by tensile shear strength test. The results suggest that DOPA and Cys in Pvfp-g and Pvfp-h cooperatively contribute to rapid protein cross-linking, enhancing the cohesive strength of the matrix of the adhesive plaque.