Article

Chemoenzymatic modification of silk fibroin with poly(2,6-dimethyl-1,5-phenylene ether) using horseradish peroxidase

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Abstract

Chemical modification of silk materials is a powerful method of tailoring the desired physical properties for possible application in various fields. In this work, we modified silk fibroin with poly(2,6-dimethyl-1,5-phenylene ether) (PPE) in order to imbue the silks with hydrophobicity so as to resist the absorption of humidity. This modification was achieved through the chemoenzymatic polymerization of 2,6-dimethylphenol (DMP) using horseradish peroxidase (HRP) as a catalyst in the presence of silk fibroin obtained from Bombyx mori. The PPE chain content in the modified silks was tuned by varying the feed concentration of DMP. Wide-angle X-ray scattering measurements revealed that β-sheet crystalline structures were formed in the PPE-modified silks, even after the introduction of bulky PPE chains. The PPE-modified silks showed glass transitions derived from the PPE domains, which enabled the formation of self-standing films upon thermal processing. Films of the PPE-modified silks exhibited higher static contact angles of water droplets compared to the native silk films, indicating that the film surface of silk fibroin became more hydrophobic because of the introduction of PPE. These improved physical properties were achieved without sacrificing the inherent secondary structure of silk fibroin, namely, the β-sheet structure that dominates the mechanical properties of silk materials.

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... A solution of the appropriate fluorinated polypeptide in ethanol (1 or 5 wt%, 60 mL) was dropped on the silk film and spin coated. Contact angle measurements on the silk films coated with a fluorinated polypeptide were performed with a DMe-201 contact angle meter (Kyowa Interface Science Co. Ltd., Saitama, Japan) [29]. A water droplet (2 mL) was placed on the film surface, and the static contact angle of the droplet was recorded after the surface tension was allowed to equilibrate for 1 s. ...
... The imparted water resistance was investigated by contact angle measurements on the silk films with fluorinated polypeptide coating. The static contact angles of water droplets were measured on the silk films coated with the different polypeptides (Fig. S2), and the results are summarized in Fig. 2. The uncoated silk film showed an average contact angle of 51.7 , which is in good agreement with the reported values [29,32]. The average contact angles of the silk films coated with P(C 3 F 7 eMe), P(C 3 F 7 -iPr), P(C 7 F 15 eMe), and P(C 7 F 15 -iPr) are slightly higher than that of the uncoated silk film, and those values raged from 57.8 to 67.4 . ...
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In this study, we prepared silkworm silk films with fluorinated polypeptide coatings on their surface to achieve water-repellent silk materials. The silk films were smoothly and uniformly coated with the fluorinated polypeptides to give a transparent film. The contact angles of water droplets on the polypeptide-coated silk films were measured to investigate the ability of the films to resist water. The silk films coated with the fluorinated polypeptides exhibited higher contact angles compared to those of the uncoated silk film, indicating that the fluorinated polypeptides formed a hydrophobic surface on the silk film. The structure of the fluorinated polypeptides influenced the water-repellent ability, and the polypeptide containing both perfluoroalkyl chains and biphenyl groups imparted the highest water repellency. A biochemical oxygen demand (BOD) assay for the polypeptide-coated silk films in seawater revealed that the silk films showed high biodegradability in seawater even with the fluorinated polypeptide coating. The polypeptide-coated silk films provide not only high water repellency for practical use but also high biodegradability in marine environments.
... The modification of silk is a facile approach to improve the interfacial adhesion properties. For instance, the tuning of silk hydrophilic/hydrophobic features was achieved via the introduction of polymer chains onto the side chains of silks [24][25][26][27][28][29], whereas the decoration of silk with catechol groups was demonstrated by mimicking the adhesion proteins that exhibit strong adhesive features toward several materials owing to the presence of this functional group in 3,4-dihydroxyphenylalanine (DOPA) residue [30][31][32]. More specifically, DOPA-modified silkworm silk from B. mori was reported to exhibit five-times higher adhesion strength for several surfaces, such as mica, polymer, and wood compared, to that of unmodified silk [30]. ...
... As the tyrosine residue in silk from B. mori mainly exists at the ends of the repetitive oligopeptide units (GAGAGY) that form a β-sheet structure, the modification of this residue would largely affect the formed structures. In fact, the slight expansion of d-spacing of βsheet crystal domains was previously confirmed in our group by introducing polyphenylene ether chains at the tyrosine residues [24], while Zhao et al. reported the structure change from random to β-sheets via the conversion of tyrosine to azobenzene units [63]. Moreover, Kaplan and coworkers reported that the decoration of silkworm silk with silica-binding peptides and SiO2 by scaffolding the tyrosine units led to an increased amount of α-helical and random coil structures [64]. ...
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Silk composites with natural rubber (NR) were prepared by mixing degummed silk and NR latex solutions. A significant enhancement of the mechanical properties was confirmed for silk/NR composites compared to a NR-only product, indicating that silk can be applied as an effective reinforcement for rubber materials. Attenuated total reflection Fourier transform infrared (ATR-FTIR) and wide-angle X-ray diffraction (WAXD) analysis revealed that a β-sheet structure was formed in the NR matrix by increasing the silk content above 20 wt%. Then, 3,4-dihydroxyphenylalanine (DOPA)-modified silk was also blended with NR to give a DOPA-silk/NR composite, which showed superior mechanical properties to those of the unmodified silk-based composite. Not only the chemical structure but also the dominant secondary structure of silk in the composite was changed after DOPA modification. It was concluded that both the efficient adhesion property of DOPA residue and the secondary structure change improved the compatibility of silk and NR, resulting in the enhanced mechanical properties of the formed composite. The knowledge obtained herein should contribute to the development of the fabrication of novel silk-based elastic materials.
... Since silk has tyrosine side chains where the phenol groups exist, this reaction is possible. In more detail, in the crosslinking mechanism, tyrosinase oxidizes the tyrosyl residues in SF with oxygen, resulting in the production of ortho-quinone residues (figure 14) [247]. Subsequently, due to the amino acids' reaction, inter-or intramolecular crosslinks are formed [248]. ...
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Due to their strong biomimetic potential, silk fibroin (SF) hydrogels are impressive candidates for tissue engineering, due to their tunable mechanical properties, biocompatibility, low immunotoxicity, controllable biodegradability, and a remarkable capacity for biomaterial modification and the realization of a specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms available for the generation of SF hydrogels (SFHs). These methods, either chemical or physical, change the structure of SF and improve its mechanical stability, although each method has its advantages and disadvantages. While chemical crosslinking agents guarantee the mechanical strength of SFH through the generation of covalent bonds, they could cause some toxicity, and their usage is not compatible with a cell-friendly technology. On the other hand, physical crosslinking approaches have been implemented in the absence of chemical solvents by the induction of β-sheet conformation in the SF structure. Unfortunately, it is not easy to control the shape and properties of SFHs when using this method. The current review discusses the different crosslinking mechanisms of SFH in detail, in order to support the development of engineered SFHs for biomedical applications.
... Since silk has the tyrosine side chains, where the phenol groups exist, this reaction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 A c c e p t e d M a n u s c r i p t 75 is possible. In more detail, in the crosslinking mechanism, tyrosinase oxidizes the tyrosyl residues in SF with oxygen, resulting in the production of ortho-quinone residues ( Figure 14) [247]. ...
Article
Silk fibroin (SF) hydrogels are impressive candidates with the strong biomimetic potential for tissue engineering due to their tunable mechanical properties, biocompatibility, low immune-toxicity, controllable biodegradability, and a remarkable capacity in the biomaterial modification and specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods will enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms to generate SF hydrogels. These methods, either chemical or physical, change SF structure and improve mechanical stability, although each method has its advantages and disadvantages. Despite chemical crosslinking agents guarantee the mechanical strength of SF hydrogel through the generation of covalent bonds, they could cause some toxicity, and their usage is not accounted for a cell-friendly technology. On the other hand, physical crosslinking approaches have implemented in the absence of chemical solvents induction β-sheets conformation in SF structure. Still, it is not easy to control the shape and properties of SF hydrogel in this method. The current review determines in more detail about different crosslinking mechanisms of SF hydrogel in order to develop engineered SF hydrogels in biomedical applications.
... Next, the hydrophilicity of the electrospun blend films increased when P(3HB-co-3HHx) was blended with SF. This is because the amide structure and the amino acids with polar side groups like serine (Ser), tyrosine (Tyr), glutamic acid (Glu) and aspartic acid (Asp) from SF improved the hydrogen bonding between water molecules and the polymer blend and thus, improved the hydrophilicity [55,56]. It has been reported that the surface hydrophilicity influences the cell adhesion and usually hydrophilic surface is preferred for cell growth [57]. ...
Article
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by microorganisms, under unbalanced growth conditions, as a carbon storage compound. PHAs are composed of various monomers such as 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Silk fibroin (SF) derived from Bombyx mori cocoons, is a widely studied protein polymer commonly used for biomaterial applications. In this study, non-woven electrospun films comprising a copolymer of 3HB and 3HHx [P(3HB-co-3HHx)], SF and their blends were prepared by electrospinning technique. The growth and osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were studied using different types of fabricated electrospun films. The differentiation study revealed that electrospun P(3HB-co-3HHx)/SF film supports the differentiation of hUC-MSCs into the osteogenic lineage, confirmed by histological analysis using Alizarin Red staining, energy dispersive X-ray (EDX) and quantitative real-time PCR analysis (qPCR). Electrospun P(3HB-co-3HHx)/SF film up-regulated the expression of osteogenic marker genes, alkaline phosphatase (ALP) and osteocalcin (OCN), by 1.6-fold and 2.8-fold respectively, after 21 days of osteogenic induction. In conclusion, proliferation and osteogenic differentiation of hUC-MSCs were enhanced through the blending of P(3HB-co-3HHx) and SF. The results from this study suggest that electrospun P(3HB-co-3HHx)/SF film is a promising biomaterial for bone tissue engineering.
... Due to the nature of silk fibroin is degraded by enzymes, controlling the availability and content of enzymes becomes very important in managing the degradation rate [87,76,85]. It has also been reported that degradation behavior can be controlled through chemical modification [88][89][90]. ...
... Subsequently, water contact angle of the film was recorded. This procedure was repeated 5 times and the average value of the contact angle was calculated (Simmons et al., 2016;Tsuchiya et al., 2019). ...
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The objective of this research was to synthesize the optimized packaging film based on polyethylene (PE), polypropylene (PP) and alkali treated-wheat straw (WS) using central composite design (CCD). In this paper, effects of three independent factors viz. polyethylene (1.8–1.08 g), polypropylene (1.2–0.72 g), and alkali treated-wheat straw (0–1.2 g) on three dependent variables viz. tensile strength, elongation at break (%) and water vapor transmission rate (WVTR) of packaging film were investigated. Response surface methodology (RSM) provided the combination for an optimum film, which was 1.8 g of polyethylene, 1.2 g of polypropylene and 1.192 g of alkali treated-wheat straw giving 45.018 MPa tensile strength, 120% elongation limit and 51.740 g/m⁻²day⁻¹ water vapor transmission rate, respectively for packaging application. The film prepared using the optimized results further characterized using several characterization techniques such as scanning electron microscopy (SEM), X-ray diffraction (XRD), mechanical testing, contact angle, water vapor transmission rate and optical characteristics test. This result demonstrated the perfect use of treated-agro-waste in a polymer matrix for green packaging application.
... For example, Burke et al. functionalized side chains with catechol to tune the adhesive properties [28]. The modification of silk with poly(2,6-dimethyl-1,5-phenylene ether) has also been demonstrated and enables tuning of the glass transition and hydrophobicity of silk [36]. Chemical modification is not limited to attaching organic motifs. ...
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Silk fibers show high toughness, ductility, biocompatibility, and biodegradability due to their high-order structure. Silk materials are not limited to native fibers and also include regenerated silk materials, such as chemically modified silk materials, composite materials, and silk-inspired artificial materials synthesized through chemoenzymatic polymerization. Here, we present the concepts and methodologies associated with the different types of silk-based materials for a wide range of fields including bioengineering and medicine and explore their versatile functionality and physical properties related to their primary and secondary structures. In this review, we will reveal the characteristics of silk materials as well as research prospects for the future.
... 16,17 Enzymatic modification is also an attractive approach because the highly selective modification of complicated compounds can be achieved under mild conditions. 18−21 The introduction of poly(2,6-dimethylenephenylene ether) onto the side chain of SF via the grafting-from method using horseradish peroxidase as a catalyst has been developed in our group to impart hydrophobicity to silk materials, 19 while Freddi and co-workers reported the tyrosinase-catalyzed modification of SF to graft the polysaccharide chitosan. 20,21 In addition, the fabrication of silk composites with other materials such as polymers 22−25 and inorganics provided the desired properties. ...
Article
Silk fibroin (SF) is a fascinating natural biomaterial that exhibits remarkable mechanical properties and biocompatibility. Meanwhile, biological adhesive materials have gathered much attention as biomedical and eco-friendly material due to their characteristic properties. Herein, we report the excellent adhesive function of enzymatically modified SF. The tyrosine residues of SF were successfully converted to dihydroxy-L-phenylalanine (DOPA) unit using tyrosinase as a biocatalyst. The content of DOPA was evaluated by amino acid composition analysis. Adhesive functions of DOPA-modified SF (DOPA-SF) between several material surfaces including mica, paper, polypropylene, wood and silk film were elucidated by tensile tests. Fourier transform infrared measurements demonstrated that the adhesion strength of DOPA-SF was assisted by the formation of a -sheet structure of silk molecules. This eco-friendly and facile method offers a new perspective to fabricate natural adhesive materials for various application areas.
... Since silk has the tyrosine side chains, where the phenol groups exist, this reaction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 A c c e p t e d M a n u s c r i p t 75 is possible. In more detail, in the crosslinking mechanism, tyrosinase oxidizes the tyrosyl residues in SF with oxygen, resulting in the production of ortho-quinone residues ( Figure 14) [247]. ...
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Bombyx mori silk fabrics were chemically modified by EPSIB (a multifunctional silicone-containing epoxy crosslinking agent). The reactivity of the epoxy groups with silk fibroin was studied by using amino acid analysis. The physical properties of the modified silks such as resiliency (both wet and dry), moisture regain, dyeing behaviors, and solubility in a mixture solvent (C2H5OH × CaCl2 : H2O = 2 : 1 : 8, molar ratio) were examined. The modified silk fabrics exhibited a significantly improved resiliency, a small increase in moisture regain and whiteness, and a slightly decreased tensile strength. The contents of Serine, Trosine, Lysine, and Histidine decreased linearly as the wet crease recovery angle (CRA) increased. The solubility in the mixture solvent also decreased as the wet CRA increased. The changes of physical properties, especially the wet CRA, were mainly due to the presence of stable cross-links between silk fibroin and epoxy groups. The DSC and TGA analyses showed that EPSIB-modified silk fibroin had a higher thermal stability compared with the control. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 91: 3579–3586, 2004
Article
The chemical reactivity of epoxide molecules toward silk fibroin was investigated by determining the rate of conversion of reactive amino acid residues. Significant differences were found between two different bifunctional epoxides, diglycidyl ethers of ethylene glycol (E) and resorcinol (R), the former reacting at a higher extent with arginine and tyrosine. The moisture regain decreased by reaction with epoxides, at a variable rate and extent, according to the hydrophobic/hydrophilic properties of epoxides. A two-step behavior was observed when moisture regain values relating to the silk content in modified silk, fibers were plotted as a function of the weight gain. Dynamic mechanical data showed that the major loss peak became broader and its temperature shifted to lower values following the increase of weight gain. The loss peak temperatures showed a linear relationship with the amount of weight gain. The fine structural changes induced by reaction with eposides will be discussed in terms of chemical and steric factors of the epoxides, as well as of epoxide location within the different structural domains of silk fibers. © 1994 John Wiley & Sons, Inc.
Article
New thermosetting poly(2-allyl-6-methylphenol-co-2,6-dimethylphenol)s (3) have been developed by oxidative coupling copolymerization of 2-allyl-6-methylphenol (1) with 2,6-dimethylphenol (2), followed by thermal curing. Copolymerization was conducted in nitrobenzene in the presence of copper(I) chloride and pyridine as the catalyst under a stream of oxygen, producing high molecular weight copolymers (Mn∼50,000) with broad molecular weight distributions (Mw/Mn∼35). The structure of resulting copolymers 3 was characterized by IR, 1H, and 13C NMR spectroscopy. Cross-linking reactions of copolymers were carried out by thermal treatment in the absence or presence of a peroxide (3 wt%, 2,5-dimethyl-2,5-di(tert-butylperoxy)-3-butane). The 10% weight loss and glass transition temperatures of the cured copolymers were 436 °C in nitrogen and 235 °C, respectively after curing at 70 °C for 1 h and 300 °C for 1 h. The average refractive index of the cured copolymer (3b) film was 1.5407, from which the dielectric constant (ε) at 1 MHz was estimated as 2.6. The ε and dissipation factor of copolymer-films at 1 MHz were directly measured from their capacitances as 2.5–2.6 and 0.0015–0.0019, respectively.
Article
The regenerated silk fibers with high strength and high biodegradability were prepared from the aqueous solution of Bombyx mori silk fibroin from cocoons with wet spinning method. Although the tensile strength of the regenerated silk fibroin fiber, 210 MPa is still half of the strength of native silk fiber, the diameter of the fiber is about 100 μm which is suitable for monofilament of suture together with high biodegradability. The high concentration (30%, w/v) of the aqueous solution of the silk fibroin which corresponds to the high concentration in the middle silkgland of silkworm was obtained. This was performed by adjusting the pH of the aqueous solution to 10.4 which corresponds to pKa value of the OH group of Tyr residues in the silk fibroin. The mixed solvent, methanol/acetic acid (7:3 in volume ratio) was used as coagulant solvent for preparing the regenerated fiber. The structural change of silk fibroin fiber by stretching was monitored with both 13C solid state NMR and X-ray diffraction methods, indicating that the high strength of the fiber is related with the long-range orientation of the silk fibroin chain with β-sheet structure.
Article
Poly(ethylene glycol) (PEG)-silk fibroin (SF) conjugates (PEG2-SF) were prepared by the chemical modification of solubilized SF with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine (actPEG2) in borate buffer at 37 °C. The IR spectra and DSC curves of PEG2-SF and SF suggested the introduction of PEG into SF by the modification and the β-sheet structure of both SF and PEG2-SF induced by the treatment with methanol aqueous solutions. The content of the PEG component in PEG2-SF was evaluated to be 67% by weight from the melting enthalpy change of PEG observed on the DSC thermogram of PEG2-SF. Water content and contact angle measurements of SF before and after the modification indicated that the hydrophilicity of the PEG2-SF surface increased compared with that of SF. The attachment and growth of fibroblast cells (L-929) on the matrix of PEG2-SF were studied by a cell culture method. PEG2-SF exhibited very low cell attachment and growth, though SF exhibited high cell attachment and growth. The filopodium of the cells attached to PEG2-SF could not be found, and the cells aggregated to form masses in scanning electron microscopy images. These results could be explained in terms of the increased hydrophilicity of the PEG2-SF surface.
Article
Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.
Article
The objective of this study was to modify the surface of poly(D,L-lactic acid) (PDLLA) with different molecular weight of silk fibroins, and assess the effects of the modified surfaces on the functions of rat osteoblasts cultured in vitro. The properties of the modified PDLLA surface and the control one were investigated by contact angle and electron spectroscopy for chemical analysis (ESCA). The former indicated the variation of hydrophilicity and the latter suggested that the modified PDLLA film using silk fibroin is enriched with nitrogen atoms. The biocompatibility of the PDLLA film may be altered and in turn affects the seeded cell functions. Therefore, attachment and proliferation of osteoblasts seeded on the modified PDLLA films and the control one were examined. Cell morphologies on these films were studied by scanning electron microscopy (SEM) and cell viability was evaluated by MTT assay. In addition, differentiated cell function was assessed by measuring the alkaline phosphatase (ALP) activity. These results suggest that the silk fibroin-modified PDLLA surface can improve the interaction between osteoblasts and the PDLLA films.
Article
Oxidative polymerization of bisphenolic monomers has been performed using peroxidase as catalyst in an aqueous organic solvent. Peroxidase induced the polymerization of an industrial product, bisphenol F, consisting of 2,2'-, 2,4'-, and 4,4'-dihydroxydiphenylmethanes. Under the selected conditions, the quantitative formation of the polymer was observed. Among the isomers, 2,4'- and 4,4'-dihydroxydiphenylmethanes were polymerized to give the polymer in high yields, whereas no polymerization of the 2,2'-isomer occurred. These data suggest that the radical transfer reaction between a phenoxy radical of the enzymatically polymerizable monomer and the enzymatically nonpolymerizable monomer frequently took place during the polymerization. Various 4,4'-dihydroxyphenyl compounds were also polymerized by peroxidase catalyst. The polymerization behaviors, and solubility and thermal properties of the resulting polymers strongly depended on the bridge structure as well as the enzyme origin. Polymers from dihydroxydiphenylmethanes showed relatively high thermal stability.
Article
Silk from the silkworm, Bombyx mori, has been used as biomedical suture material for centuries. The unique mechanical properties of these fibers provided important clinical repair options for many applications. During the past 20 years, some biocompatibility problems have been reported for silkworm silk; however, contamination from residual sericin (glue-like proteins) was the likely cause. More recent studies with well-defined silkworm silk fibers and films suggest that the core silk fibroin fibers exhibit comparable biocompatibility in vitro and in vivo with other commonly used biomaterials such as polylactic acid and collagen. Furthermore, the unique mechanical properties of the silk fibers, the diversity of side chain chemistries for 'decoration' with growth and adhesion factors, and the ability to genetically tailor the protein provide additional rationale for the exploration of this family of fibrous proteins for biomaterial applications. For example, in designing scaffolds for tissue engineering these properties are particularly relevant and recent results with bone and ligament formation in vitro support the potential role for this biomaterial in future applications. To date, studies with silks to address biomaterial and matrix scaffold needs have focused on silkworm silk. With the diversity of silk-like fibrous proteins from spiders and insects, a range of native or bioengineered variants can be expected for application to a diverse set of clinical needs.
Article
High-resolution solution (13)C-NMR and CD studies of Bombyx mori silk fibroin revealed the presence of an ordered secondary structure 3(10)-helix, in hexafluoro-iso-propanol (HFIP). The solid-state structure of the silk fibroin film prepared by drying it gently from the HFIP solution still keep the structure, 3(10)-helix, which was studied with high-resolution solid state (13)C-NMR. The structural transition from the 3(10)-helix to silk II structure, heterogeneous structure including antiparallel beta-sheet, occurred during the artificial spinning from the HFIP solution. The wide-angle x-ray diffraction and differential scanning calorimetry thermograms of the artificial spinning fiber after postspinning treatments were observed together with the stress-strain curves. The results emphasize that the molecular structures, controlled morphology, and mechanical properties of the protein-based synthetic polymers can be modulated for enhancing biocompatibility.
Article
The potential for using tyrosinase to graft the polysaccharide chitosan (Ch) onto Bombyx mori silk fibroin (SF) was examined. FT-IR spectroscopy coupled to HPLC amino acid analysis showed that mushroom tyrosinase (MT) catalyses the oxidation of tyrosine (Tyr) of SF to electrophilic o-quinones. Kinetic studies showed that only a fraction of the Tyr residues available on the SF chain were oxidized. This result was interpreted in the light of the structure assumed by SF in aqueous solution: Tyr aromatic side chain groups buried into the folded hydrophobic portions of the chain were probably less accessible to MT for steric reasons. Using slightly acidic conditions (pH 6), it was possible to modify SF under homogeneous conditions. FT-IR spectroscopy provided evidence that Ch was grafted onto MT-oxidized SF: the o-quinones were found to undergo a subsequent non-enzymatic reaction with nucleophilic amino groups of Ch via Schiff-base and Michael addition mechanisms. Various factors, i.e. reaction time, pH, MT/SF ratio, were found to influence the grafting yield. The highest grafting yield was achieved at pH 7, i.e. more favorable to MT activity rather than to Ch solubility, suggesting that the determining step of the grafting reaction is the formation of o-quinones. The FT-IR spectroscopy revealed that grafting induced a beta-sheet --> random coil conformational transition.
Article
Horseradish peroxidase is an important heme-containing enzyme that has been studied for more than a century. In recent years new information has become available on the three-dimensional structure of the enzyme and its catalytic intermediates, mechanisms of catalysis and the function of specific amino acid residues. Site-directed mutagenesis and directed evolution techniques are now used routinely to investigate the structure and function of horseradish peroxidase and offer the opportunity to develop engineered enzymes for practical applications in natural product and fine chemicals synthesis, medical diagnostics and bioremediation. A combination of horseradish peroxidase and indole-3-acetic acid or its derivatives is currently being evaluated as an agent for use in targeted cancer therapies. Physiological roles traditionally associated with the enzyme that include indole-3-acetic acid metabolism, cross-linking of biological polymers and lignification are becoming better understood at the molecular level, but the involvement of specific horseradish peroxidase isoenzymes in these processes is not yet clearly defined. Progress in this area should result from the identification of the entire peroxidase gene family of Arabidopsis thaliana, which has now been completed.
Article
Horseradish peroxide (HRP) was covalently coupled to three-dimensional (3D) silk fibroin scaffolds using water-soluble carbodiimide. Stable, bilaterally symmetrical immobilized HRP gradient patterns were generated within 3D silk fibroin scaffolds using the principles of diffusion. Gradients of immobilized HRP activity were controlled using variables of volume and concentration of HRP solution activated by the carbodiimide. The method developed can be extended to immobilize a variety of proteins and small molecules on several types of porous, interconnected materials. This technique of patterning enzymes and proteins in a gradient manner offers new options in the field of chemotaxis, tissue engineering, and biosensors.
Article
The capability of mushroom tyrosinase to catalyze the oxidation of tyrosine residues of Bombyx mori silk fibroin was studied under heterogeneous reaction conditions, by using a series of silk substrates differing in surface and bulk morphology and structure, i.e. hydrated and insoluble gels, mechanically generated powder and fibre. Tyrosinase was able to oxidize 10-11% of the tyrosine residues of silk gels. The yield of the reaction was very low for the powder and undetectable for fibres. FT-Raman spectroscopy gave evidence of the oxidation reaction. New bands attributable to vibrations of oxidized tyrosine species (o-quinone) appeared, and the value of the I853/I829 intensity ratio of the tyrosine doublet changed following oxidation of tyrosine. The thermal behaviour of SF substrates was not affected by enzymatic oxidation. o-Quinones formed by tyrosinase onto gels and powder were able to undergo non-enzymatic coupling with chitosan. FT-IR and FT-Raman spectroscopy provided clear evidence of the formation of silk-chitosan bioconjugates under heterogeneous reaction conditions. Chitosan grafting caused a beta-sheet --> random coil conformational transition of silk fibroin and significant changes in the thermal behaviour. Chitosan grafting did not occur, or occurred at an undetectable level on silk fibres. The results reported in this study show the potential of the enzymatically initiated protein-polysaccharide grafting for the production of a new range of bio-based, environmentally friendly polymers.
Article
The thermal behavior up to degradation of Bombyx mori silk has been studied by scanning synchrotron radiation microdiffraction, gel electrophoresis, and mechanical testing. The diffraction patterns from single baves can be separated into scattering from anisotropic crystalline beta-sheet domains and random short-range order. In contrast to dragline silk, scattering from oriented, short-range-order fibroin is not observed. The sheath of sericin proteins can be selectively probed by a microbeam and shows also principally random short-range-order domains with a small crystalline beta-sheet fraction. Microdiffraction experiments on single baves from 100 to 573 K show an increase in lattice expansion along the [010] chain-stacking direction above 200-250 K, which could be due to an increase in side-chain mobility. Degradation of the crystalline fraction commences at approximately 500 K, and the fibers have become amorphous at about 570 K with an onset of carbonization. Gel electrophoresis shows that the degradation of FibH molecules starts already at about 350 K, while FibL molecules start degrading at about 400 K. The mechanical properties of single baves such as strain-to-failure and tensile strength also start degrading at about 400 K, while the initial modulus increases up to about 475 K. It is proposed that this is due to the development of cross-linking in the short-range-order chain fraction.
Article
Polymers produced by horseradish-peroxidase-catalyzed coupling of phenols have been explored as potential substitutes for phenol-formaldehyde resins. To overcome low substrate solubilities and product molecular weights in water, enzymatic polymerizations in aqueous-organic mixtures have been examined. Peroxidase vigorously polymerizes a number of phenols in mixtures of water with water-miscible solvents such as dioxane, acetone, di-methylformamide, and methyl formate with the solvent content up to 95%. As a result, various phenolic polymers with average molecular weights from 400 to 2.6 x 10(4) D were obtained depending on the reaction medium composition and the nature of the phenol. Peroxidase-catalyzed copolymerization of different phenols in 85% dioxane was demonstrated. Poly(p-phenylphenol) and poly(p-cresol) were enzymatically prepared on a gram scale. They had much higher melting points, and in addition, poly(p-phenylphenol) was found to have a much higher electrical conductivity than phenol-formaldehyde resins.
Article
Phenolic polymerization was carried out by enzymatic catalysis in organic media, and its kinetics was studied by using high-pressure liquid chromatography (HPLC). Phenols and aromatic amines with electron-withdrawing groups could hardly be polymerized by HRP catalysis, but phenols and aromatic amines with electron-donating groups could easily be polymerized. The reaction rate of either the para-substituted substrate or meta-substituted substrate was higher than that of ortho-substituted substrate. When ortho-position of hydroxy group of phenols was occupied by an electron-donating group and if another electron-donating group occupied para-position of hydroxy group, the reaction rate increased. Horseradish peroxidase and lactoperoxidase could easily catalyze the polymerization, but chloroperoxidase and laccase failed to yield polymers. Metallic ions such as Mn(2+), Fe(2+), or Fe(3+), and Cu(2+) could poison horseradish peroxidase to various extents, but ions such as Co(2+), Cd(2+), Zn(2+), and K(+) were not found to inhibit the reaction. (c) 1995 John Wiley & Sons, Inc.
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