Nucleation of hydroxyapatite crystals by self-assembled Bombyx mori silk fibroin

Bombyx mori silk fibroin (SF) is known to be capable of facilitating nucleation of the hydroxyapatite crystals (HAps). To find out how SF mediates the nucleation of HAps, self-assembly of SF in 1.5 simulated body fluid (SBF) was observed in this study through design of a co-solution of SF and 1.5-times SBF (SF/1.5 SBF). After the co-solution of SF/1.5 SBF was incubated at 37.2 °C up to 7 days, SEM, X-ray, and Fourier transform infrared (FTIR) observations indicated that nucleation of HAps was increased. In addition, the structure of SF was transited from random coil into β-sheet indicated by FTIR spectra. The β-sheet assembly of SF in 1.5 SBF was also supported by CD spectra. Atomic force microscopy provided detailed progress of the self-assembly that SF incubated in 1.5 SBF was self-assembled in the form from dot, through rod to final net. Therefore, this study suggested that nucleation of HAps of SF was controlled by its molecular self-assembly. © 2013 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2013     

Dissolution and regeneration of Bombyx mori silk fibroin using ionic liquids

In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering. The dissolved silk is also processed into 100 mum-thick, two-dimensional films, and the structure of these films is examined. The rinse solvent, acetonitrile or methanol, has a profound impact on both the topography of the films and the secondary structure of the silk protein. The image depicts a silkworm cocoon dissolved in 1-butyl-3-methylimidazolium chloride and then regenerated as a film with birefringence.     

Effect of metallic ions on silk formation in the Mulberry silkworm, Bombyx mori

A protein conformation transition from random coil and/or helical conformation to beta-sheet is known to be central to the process used by silk-spinning spiders and insects to convert concentrated protein solutions to tough insoluble threads. Several factors including pH, metallic ions, shear force, and/or elongational flow can initiate this transition in both spiders and silkworms. Here, we report the use of proton induced X-ray emission (PIXE), inductively coupled plasma mass spectroscopy (ICP-MS) and atomic adsorption spectroscopy (AAS) to investigate the concentrations of six metal elements (Na, K, Mg, Ca, Cu, and Zn) at different stages in the silk secretory pathway in the Bombyx mori silkworm. We also report the use of Raman spectra to monitor the effects of these six metallic ions on the conformation transition of natural silk fibroin dope and concentrated regenerated silk fibroin solution at concentrations similar to the natural dope. The results showed that the metal element contents increased from the posterior part to the anterior part of silk gland with the exception of Ca which decreased significantly in the anterior part. We show that these changes in composition can be correlated with (i) the ability of Mg2+, Cu2+, and Zn2+ to induce the conformation transition of silk fibroin to beta-sheet, (ii) the effect of Ca2+ in forming a stable protein network (gel), and (iii) the ability of Na+ and K+ to break down the protein network.     

Self-assembly of a peptide amphiphile based on hydrolysed Bombyx mori silk fibroin

An octapeptide, GAGAGAGY, was obtained by a novel method, i.e. hydrolysing Bombyx mori silk fibroin. Afterward, a dodecanoic acid-peptide conjugation was synthesized. This amphiphile assembled into cylindrical nanofibers of planar β-sheets at pH 9 and twisted β-sheets at pH 4.     

Microstructure elucidation of historic silk (Bombyx mori) by nuclear magnetic resonance

¹H NMR cryoporometry and solid-state ¹³C cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy were used to characterize the microstructure of historic and fresh silk samples. Silk is a polymeric bicomponent material composed of fibroin and water located in micropores. According to the ¹H NMR cryoporometry method, the intensity of the water resonance as a function of the temperature was used to obtain the pore size distribution, which was strongly asymmetric with a well-defined maximum at 1.1 nm. Compared with the fresh silk samples, the volume of pores around 1.1 nm decreased distinctly in the historic silk, and more pores larger than 2 nm emerged accordingly. In addition, these results correlated well with solid-state ¹³C CP/MAS NMR spectroscopy as the percentage of random coil in the historic silk sample was much less than that in the fresh silk samples. Therefore, it is suggested that the water-filled microvoids grow larger as the random coil conformation fades away in the degradation process.

用桑蚕丝素蛋白固定脲酶的研究及脲酶电极的制备

提出了一种新的酶固定化方法, 即通过甲醇处理, 使蚕丝素蛋白膜的构象由random coil向β-sheet发生根本性的变化, 从而将酶固定在β-sheet所特有的分子间氢键中。利用此方法所制成的脲酶电极, 在合适的操作条件下, 各项响应指标均令人满意, 并且脲酶的耐温性能被大大提高, 电极的有效使用寿命长达三个月以上。此种酶固定化方法原则上能够应用于其他不破坏蚕丝素蛋白分子结构的可溶性酶。     

Structural role of tyrosine in Bombyx mori silk fibroin, studied by solid-state NMR and molecular mechanics on a model peptide prepared as silk I and II

The influence of the bulky and H-bonding Tyr side-chain on its Ala- and Gly-rich environment in Bombyx mori silk fibroin was examined by (13)C cross-polarization magic angle spinning (CP/MAS), static (2)H and (19)F NMR and molecular mechanics calculations. Model peptides of the type (AG)(15) were synthesized with Tyr in a number of different positions, precipitated under conditions favoring either of the two characteristic protein conformations, and the resulting structures were assigned from their (13)C chemical shifts. Dialysis of native fibroin or the simple (AG)(15) peptide from a 9 M LiBr solution against water produces silk I (the structure of silk before spinning), whereas drying from formic acid yields silk II (fibrous structure after spinning). We found that the introduction one or more Tyr into (AG)(15) can have a dramatic effect not only on the local backbone conformation but also on the long-range intermolecular chain packing in the samples. The antiparallel beta-sheet conformation of silk II is able readily to accommodate a single Tyr residue. Interestingly, the beta-turn conformation of silk I only remains stable when Tyr is positioned near the chain terminus in (AG)(12)YG(AG)(2), but the conformation is driven towards silk II when Tyr is located in the central region of (AG)(7)YG(AG)(7). The role of H-bonding was tested by replacing Tyr with Phe or 4F-Phe, which are no longer compatible with silk I and fully induced a silk II conformation. In the presence of several Tyr residues a mixture of distorted beta-sheet and beta-turn conformations was obtained, regardless of the precipitation conditions. Static (2)H NMR of ring-deuterated [3',5'-(2)H(2)]Tyr located in the central region of (AG)(7)YG(AG)(7) showed that the side-chain is immobilized in both silk I and II, which was also observed by static (19)F NMR of the 4F-Phe analogue. To visualize the local packing around the Tyr side-chain, molecular mechanics calculations were performed on a mixture of (AG)(4) and AGAGYGAG, starting from either the beta-turn type II or the antiparallel beta-sheet structure. The resulting structures show that the intermolecular chain arrangement is significantly affected by Tyr, thus explaining the long-range packing effects in the semi-crystalline regions of silk fibers compared with the crystalline regions that are devoid of Tyr.     

Atomic force microscopy: Bombyx mori silk fibroin molecules and their higher order structure

We performed atomic force microscopy measurements on fibroin molecules from the domestic silkworm, Bombyx mori. At low concentrations, we could observe single protein molecules. The shape of the observed molecules is a rod with long smaller chains extending from the ends. The size of the rod is 60 nm in length and 15 nm in width. At high concentrations, we observed long threadlike aggregates of fibroin molecules, their chains entangled with one another. These results provide insight into the microscopic mechanism of silk‐fiber formation. © 2000 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 38: 1436–1439, 2000     

Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy

The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.     

Influence of an iodine treatment on the structure and physical properties of Bombyx mori silk fibroin fiber

Silk fibroin (SF) fiber from the Bombyx mori silkworm was treated with a 1.23 N iodine/potassium iodide (I₂–KI) aqueous solution, and the structure and physical properties were investigated to elucidate the effects of the iodine treatment. The SF fiber absorbed polyiodide ions such as I and I by immersion in the I₂–KI solution, and the weight gain of the SF fiber increased with the treatment time; it became saturated at about 20 wt % after 40 h. The results of the weight gain, Fourier transform infrared spectroscopy, and X‐ray diffraction measurements suggested that polyiodide ions mainly entered the amorphous region. Moreover, a new sharp reflection in the meridional direction, corresponding to a period of 7.0 Å, was observed and indicated the possibility of the formation of a mesophase structure of β‐conformation chains. Dynamic viscoelastic measurements showed that the molecular motion of the crystalline regions at about 220 °C was enhanced and shifted to lower temperature by the introduction of polyiodide ions. This indicated that the iodine component weakened the hydrogen bonding between the SF molecules forming the β‐sheet structure and caused molecular motion of the crystal to occur more easily with heating. With heating above 270 °C, the iodine component introduced intermolecular crosslinking to SF, and the melt flow of the sample was inhibited. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 3418–3426, 2006     

Physical properties and phase separation structure of Antheraea pernyi/Bombyx mori silk fibroin blend films

The physical structure and compatibility of solution-cast Antheraea pernyi/Bombyx mori silk fibroin blend films were stuided by differential scanning calorimetry (DSC), thermomechanical (TMA) and thermogravimetric (TGA) analysis, dynamic viscoelastic measurement, infrared spectroscopy, and x-ray diffractometry. The DSC curves of the blend films showed independent endotherms at 280 and 358°C, corresponding to the thermal decomposition of B. mori and A. pernyi silk fibroins with random coil conformation. The intensity was roughly proportionate to the amount of each component in the blend. The thermal behavior corresponding to the conformational transitions induced by heating on A. pernyi and B. mori silk fibroins overlapped in the temperature range 190–230°C. Thermal expansion and contraction properties, as well as weight retention behavior of the blend films were intermediate between the pure components, as shown by the TMA and TGA curves. The onset temperature of the storage modulus curve decreased markedly, approaching that of B. mori silk fibroin film when the amount of this component in the blend increased. The loss modulus curve of the blend films showed two peaks at ca. 190 and 210°C, the former corresponding to B. mori, and the latter to A. pernyi silk fibroin. Infrared spectra of the blends exhibited absorption bands characteristic of the pure components overlapping in the spectral region 2000–400 cm^?1. The x-ray diffraction peaks at 23 and 21.5°, attributed to the crystalline spacings of A. pernyi and B. mori fibroins, respectively, overlapped in the diffraction curves of the blends, while the peak at 11.4°, of A. pernyi, increased as the content of this fibroin in the blend increased. The degree of crystallinity, calculated from the x-ray diffraction curves, diminished as the amount of B. mori silk fibroin decreased. A low degree of compatibility exists between the two fibroins when they are cast from aqueous solution in the experimental conditions adopted in this work. © 1994 John Wiley & Sons, Inc.     

Heterologous expression characteristics of Trichoderma viride endoglucanase V in the silkworm, Bombyx mori L

Efficient degradation of cellulose needs a synergistic reaction of the cellulolytic enzymes, which include exoglucanases, endoglucanases, and β-1,4-glucosidase. In this study, we used an improved Bac-to-Bac/BmNPV baculovirus expression system, which lacks the virus-encoded chitinase cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV), to express the endoglucanase V (EG V) gene from Trichoderma viride in silkworm BmN cells and silkworm larvae, and analyzed the characteristics of the recombinant enzyme in silkworm larvae. The result showed that an around 36-kDa protein was visualized in BmN cells at 48 h after the second-generation recombinant mBacmid/BmNPV/EG V baculovirus infection. The crude enzyme extract from the recombinant baculoviruses-infected silkworms exhibited a significant maximum activity at the environmental condition of pH 5.0 and a temperature of 50 °C, and increased 39.86% and 37.76% compared with that from blank mBacmid/BmNPV baculovirus-infected silkworms and normal silkworms, respectively. It was stable at pH range from 5.0 to 10.0 and at temperature range from 40 to 60 °C. The availability of large quantities of EG V that the silkworm provides might greatly facilitate the future research and the potential application in industries.     

Zur Bestimmung von Magnesium im Polyeder-Virus und Polyeder-Protein von Bombyx mori L

Ohne Zusammenfassung     

Water dependent structural changes of silk from Bombyx mori gland to fibre as evidenced by Raman and IR spectroscopies

DSC, attenuated total reflexion infrared (ATR-FTIR), and Raman microspectroscopy were used for the first time in a close to in vivo environment to study ready-to-spin Bombyx mori silkworms. The aim was to understand the change of the fibroin backbone organisation from the gland to the fibre. Raman shifts of the Amide I components reveal a strong change of organisation in the middle part of the hydrated gland, as anticipated previously measured modifications of salts concentrations and pH. Series of bands characteristics of the fully hydrated silk disappear, as observed for spider silk, despite the different aminoacid sequence. Confirmation is obtained from IR spectra taking into account the superimposed water component. The change of the silk–water interaction in the central part of the gland, from a hydrophobic to hydrophilic behaviour, is related to the water content decrease along the gland. pH sensitive carboxylate side chains markers confirm the modification. Fibroin organisation was also studied in the dried gland and in the spun fibre. The fibre extrusion by orients the fibroin chains along the fibre axis, with intercalated water molecules, leading to a material with specific mechanical properties, compared to the amorphous dried gland.     

Wax components of larval cocoon silk of the hornet Vespa analis Fabricius

Wax, 85% of which consists of orthorhombic crystals, has been found in the cocoon of the hornet Vespa analis Fabricius by means of high-resolution ¹³C solid-state nuclear magnetic resonance (NMR). GC–MS analysis revealed the major components of the wax in the cocoon were linear alkenes and alkanes with a total of 23 or 34 carbon atoms. At 40.7 °C a DSC absorption peak and a ¹³C NMR chemical shift change were observed and interpreted as the result of a crystal transition from the orthorhombic to rotator phase of the wax molecules. At 55.5 °C melting of the wax was observed. The amount of crystalline wax deposition varied with the part of the cocoon—crystalline wax was concentrated in the silk sleeve lining the inner wall of each comb cell but there was very little in the silk cap projecting from the end of each cell. Because the wax components of the larval cocoon were almost identical to those of the larval cuticle, despite a slight difference in the profiles, they might have come from the larval cuticle via direct body contact with the cocoon. Figure Cocoon of the hornet Vespa analis Fabricius