Tyrosinase-catalyzed grafting of sericin peptides onto chitosan and production of protein-polysaccharide bioconjugates

The capability of Agaricus bisporus tyrosinase to catalyze the oxidation of tyrosine residues of silk sericin was studied under homogeneous reaction conditions, by using sericin peptides purified from industrial wastewater as the substrate. Tyrosinase was able to oxidize about 57% of sericin-bound tyrosine residues. The reaction rate was higher than with silk fibroin, but lower than with other silk-derived model peptides, i.e. tryptic and chymotryptic soluble peptide fractions of silk fibroin, suggesting that the size and the molecular conformation of the substrate influenced the kinetics of the reaction. The concentration of tyrosine in oxidized sericin samples decreased gradually with increasing the enzyme-to-substrate ratio. The average molecular weight of sericin peptides significantly increased by oxidation, indicating that cross-linking occurred via self-condensation of o-quinones and/or coupling with the free amine groups of lysine and, probably, with sulfhydryl groups of cysteine. The high temperature shift of the main thermal transitions observed in the differential scanning calorimetry curves confirmed the formation of peptide species with higher molecular weight and higher thermal stability. Fourier transform-infrared spectra of oxidized sericin samples showed slight changes related to the loss of tyrosine and formation of oxidation products. Oxidized sericin peptides were able to undergo non-enzymatic coupling with chitosan. Infrared spectra provided clear evidence of the formation of sericin-chitosan bioconjugates under homogeneous reaction conditions. Spectral changes in the NH stretching region seem to support the formation of bioconjugates via the Michael addition mechanism.     

酪氨酸酶催化氧化对丝素蛋白结构与性能的影响

采用酪氨酸酶对丝素蛋白催化氧化,考察了酶促氧化反应对丝素蛋白结构及丝素膜性能的影响。研究结果表明,酪氨酸酶可催化氧化丝素蛋白中酪氨酸残基生成多巴和多巴醌结构衍生物,并且两者含量随催化反应时间延长呈波动性变化;酶促反应后丝素蛋白中游离氨基含量下降,丝素风干膜断裂强度增加,表明酶促氧化中丝素大分子间发生自交联。XRD结果表明酪氨酸酶催化氧化对丝素蛋白二级结构有一定影响;SEM显示酶促改性可能影响丝素蛋白冷冻干燥膜多孔形态结构。     

Modification of Bombyx mori silk fabrics by tyrosinase‐catalyzed grafting of chitosan

Tyrosinase could oxidize tyrosyl residues in silk fibroin and result in the production of activated o‐quinone residues, which could facilitate the grafting of the functional amino‐compounds onto silk fibers. In this study, the enzymatic modifications of Bombyx mori silk fibroin with tyrosinase and chitosan were investigated, aiming at improving the properties of silk fabrics, including dyeability, crinkling resistance, and antibacterial activity. The grafting grades of chitosan were evaluated by a color‐development method using bromocresol green. The result indicated that chitosan molecules were not only adsorbed on silk fibers via electrostatic interactions, they also could react with the oxidized silk fibers with tyrosinase. For the silk fabric combinedly treated with tyrosinase and chitosan, tensile strength and crinkling resistance were noticeably increased as compared to that of the chitosan‐treated. The antibacterial activity and its durability measurements revealed the actions of the tyrosinase‐catalyzed grafting of chitosan. The efficacy of the graft reaction might be further enhanced by increasing the accessibility of reactive sites in silk fibers.     

Enzymatic grafting of chitosan onto Bombyx mori silk fibroin: kinetic and IR vibrational studies

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.     

Transglutaminase mediated grafting of silk proteins onto wool fabrics leading to improved physical and mechanical properties

Transglutaminases have the ability to incorporate primary amines and to graft peptides (containing glutamine or lysine residues) into proteins. These properties enable transglutaminases to be used in the grafting of a range of compounds including peptides and/or proteins onto wool fibres, altering their functionality. In this paper we investigated the transglutaminase mediated grafting of silk proteins into wool and its effect on wool properties. A commercial hydrolysed silk preparation was compared with silk sericin. The silk sericin protein was labelled with a fluorescent probe which was used to demonstrate the efficiency of the TGase grafting of such proteins into wool fibres. The TGase mediated grafting of these proteins led to a significant effect on the properties of wool yarn and fabric, resulting in increased bursting strength, as well as reduced levels of felting shrinkage and improved fabric softness. Also observed was an accumulation of deposits on the surface of the treated wool fibres when monitored by SEM and alterations in the thermal behaviour of the modified fibres, in particular for mTGase/sericin treated fibres which, with the confocal studies, corroborate the physical changes observed on the treated wool fabric.     

In vitro protein-polysaccharide conjugation: tyrosinase-catalyzed conjugation of gelatin and chitosan

The enzyme tyrosinase was used for the in vitro conjugation of the protein gelatin to the polysaccharide chitosan. Tyrosinases are oxidative enzymes that convert accessible tyrosine residues of proteins into reactive o-quinone moieties. Spectrophotometric and dissolved oxygen studies indicate that tyrosinase can oxidize gelatin and we estimate that 1 in 5 gelatin chains undergo reaction. Oxidized tyrosyl residues (i.e., quinone residues) can undergo nonenzymatic reactions with available nucleophiles such as the nucleophilic amino groups of chitosan. Ultraviolet/visible, (1)H-NMR, and ir provided chemical evidence for the conjugation of oxidized gelatin with chitosan. Physical evidence for conjugation was provided by dynamic viscometry, which indicated that tyrosinase catalyzes the sol-to-gel conversion of gelatin/chitosan mixtures. The gels formed from tyrosinase-catalyzed reactions were observed to differ from gels formed by cooling gelatin. In contrast to gelatin gels, tyrosinase-generated gels had different thermal behavior and were broken by the chitosan-hydrolyzing enzyme chitosanase. These results demonstrate that tyrosinase can be exploited for the in vitro formation of protein-polysaccharide conjugates that offer interesting mechanical properties.     

Chemical and physical properties of sulfated silk fabrics

Silk fabrics were treated with chlorosulphonic acid in pyridine for different times. The amount of sulfur bound to silk increased during the first 2 h of reaction and then reached a plateau. The amino acidic pattern of sulfated silk remained essentially unchanged for short reaction times (< or =2 h). Longer reaction times resulted in drastic changes in the concentration of Asp, Glu, and Tyr. Surface morphology and texture of silk fabrics changed upon sulfation. Warp and weft yarns became progressively thinner, and deposits of foreign material appeared on the fiber surface. Changes were more evident at longer reaction times (> or =2 h). Spectroscopic analyses performed by FT-IR and FT-Raman showed the appearance of new bands attributable to various vibrations of sulfated groups. The IR bands at 1049 and 1014 cm-1, due to organic sulfate salts, were particularly intense. Bands assigned to alkyl sulfates and sulfonamides appeared in the 1300-1180 cm-1 range. Organic covalent sulfates displayed a weak but distinct IR band at 1385 cm-1. Both IR and Raman spectra revealed that silk fibroin mainly bound sulfates through the hydroxyl groups of Ser and Tyr, while involvement of amines could not be proved. Changes observed in the amide I and II range indicated an increase of the degree of molecular disorder of sulfated silk. Accordingly, the I850/I830 intensity ratio between the two Tyr bands at 850-830 cm-1 increased from 1.41 to 1.52, indicating a more exposed state of Tyr residues in sulfated silk. TGA, DSC, and TG analyses showed that sulfated silk attained a higher thermal stability. A thermal transition attributable to sulfated silk fibroin fractions appeared at about 260 degrees C in the DSC thermograms.     

Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA

L-DOPA (3,4-dihydroxyphenyl-L-alanine), one of the most important intermediates in the melanin biosynthesis pathway, is used for the treatment of Parkinson's disease. With a view of developing a cheaper and more effective method for the bioconversion of tyrosine to L-DOPA, the potential and performance of a novel fibrous matrix prepared from Bombyx mori silk protein fibroin were evaluated for the immobilization of tyrosinase. Cross-linkage between fibroin and tyrosinase using glutaraldehyde was evident from Fourier transform infra red spectroscopy. Maximum product formation occurred when 1000 U enzyme was immobilized on 20 mg fibroin. The optimum conditions for maximal L-DOPA production using immobilized tyrosinase were 40 degrees C and pH 5.5, conditions that caused a 50% loss of free enzyme activity. Immobilized tyrosinase also showed to have a higher degree of stability during storage and it retained 80% of its original activity after repeated reuses. The efficiency of this immobilized tyrosinase system to produce L-DOPA was high, as evident from a high effectiveness factor, between 0.7 and 0.8, thereby making this method feasible for the large-scale production of L-DOPA.     

13C CP/MAS NMR study on structural heterogeneity in Bombyx mori silk fiber and their generation by stretching

It is important to resolve the structure of Bombyx mori silk fibroin before spinning (silk I) and after spinning (silk II), and the mechanism of the structural transition during fiber formation in developing new silk-like fiber. The silk I structure has been recently resolved by (13)C solid-state NMR as a "repeated beta-turn type II structure." Here, we used (13)C solid-state NMR to clarify the heterogeneous structure of the natural fiber from Bombyx mori silk fibroin in the silk II form. Interestingly, the (13)C CP/MAS NMR revealed a broad and asymmetric peak for the Ala Cbeta carbon. The relative proportions of the various heterogeneous components were determined from their relative peak intensities after line shape deconvolution. Namely, for 56% crystalline fraction (mainly repeated Ala-Gly-Ser-Gly-Ala-Gly sequences), 18% distorted beta-turn, 13% beta-sheet (parallel Ala residues), and 25% beta-sheet (alternating Ala residues). The remaining fraction of 44% amorphous Tyr-rich region, 22% in both distorted beta-turn and distorted beta-sheet. Such a heterogeneous structure including distorted beta-turn can be observed for the peptides (AG)(n) (n > 9 ). The structural change from silk I to silk II occurs exclusively for the sequence (Ala-Gly-Ser-Gly-Ala-Gly)(n) in B. mori silk fibroin. The generation of the heterogeneous structure can be studied by change in the Ala Cbeta peak of (13)C CP/MAS NMR spectra of the silk fibroin samples with different stretching ratios.     

Antimicrobial and antioxidant properties of chitosan enzymatically functionalized with flavonoids

Chitosan fibres were grafted with flavonoids using tyrosinase to produce reactive o-quinones which subsequently react with primary amino groups of the chitosan. The reaction mechanism using chemically different flavonoids (flavanols, flavonols, flavone, flavanone, isoflavone) was followed by UV/vis spectroscopy and the successful grafting was demonstrated by ATR-IR spectroscopy, pH potentiometric titration and reflectance measurements. An increase of antioxidant activity of functionalized chitosan fibres using well established methods was found depending on the type of the flavonoid used. In addition, some flavonoids increased antimicrobial activity of chitosan against Bacillus subtillis and Pseudomonas aeruginosa.     

The preparation and applications of functional fibres from crab shell chitin

Novel chitin–silk fibroin fibres and chitin fibres were prepared by an environmental friendly wet-spinning method. Each aqueous solution of sodium chitin (N-acetylchitosan) salt and its blends of silk fibroin in aqueous 14% sodium hydroxide was spun through a viscose-type spinneret into an aqueous 10% sulfuric acid solution saturated with ammonium sulfate (about 43%), and the corresponding white filament was obtained. The tenacity and elongation values of the chitin–silk fibroin filament decreased with an increase of fibroin content up to 33% by weight. A scanning electron microscopy analysis revealed that both the chitin filament and the chitin–silk fibroin (67:33, w/w) filament had vertical strips with faint scale structures on their surfaces. Some applications of these staple fibres were also reported.     

Silk cocoon of Bombyx mori: Proteins and posttranslational modifications – heavy phosphorylation and evidence for lysine‐mediated cross links

Although silk is used to produce textiles and serves as a valuable biomaterial in medicine, information on silk proteins of the cocoon is limited. Scanning electron microscopy was applied to morphologically characterise the sample and the solubility of cocoon in lithium thiocyanate and 2‐DE was carried out with multi‐enzyme in‐gel digestion followed by MS identification of silk‐peptides. High‐sequence coverage of the silk cocoon proteins fibroin light and heavy chain, sericins and fibrohexamerins was revealed and PTMs as heavy phosphorylation of silk fibroin heavy chain; lysine hydroxylation and Lys‐>allysine formation have been observed providing evidence for lysine‐mediated cross linking of silk as found in collagens, which has not been reported so far. Tyrosine oxidation verified the presence of di‐tyrosine cross links. A high degree of sequence conflicts probably representing single‐nucleotide polymorphisms was observed. PTM and sequence conflicts may be modulating structure and physicochemical properties of silk.     

Preparation, characterization and biocompatibility of electrospinning heparin-modified silk fibroin nanofibers

In this study, the electrospun silk fibroin nanofibrous scaffolds were modified with heparin by grafting after plasma treatment and blending electrospinning. Morphology, microstructure, chemical composition and grafting efficiency of the heparin-modified silk fibroin nanofibrous scaffolds were characterized to evaluate the effect of modification by means of scanning electron microscopy (SEM), Fourier transform infrared spectra (FTIR) and X-ray photoelectron spectrometer (XPS). The results showed that the heparin was successfully introduced to the silk fibroin nanofibrous scaffolds by both the two kinds of modification, and there was a hydrogen bonding between the silk fibroin and heparin. Moreover, the hydrophilicity, O-containing groups and negative charge density of the heparin-modified scaffolds were enhanced. In vitro coagulation time tests showed that the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) of the heparin-modified scaffolds were much higher than those of the pure silk fibroin scaffolds. L929 fibroblasts and EVCs spread and proliferated better on the heparin-modified scaffolds than on the pure silk fibroin scaffolds. Macrophages, neutrophils and lymphocytes were not observed in the heparin-modified scaffolds, which indicated that the modified scaffolds could induce minor inflammation in vivo. The results indicated that the electrospun heparin-modified silk fibroin nanofibrous scaffolds could be considered as ideal candidates for tissue engineering scaffolds.     

Surface Modification and Characterisation of Silk Fibroin Fabric Produced by the Layer-by-Layer Self-Assembly of Multilayer Alginate/Regenerated Silk Fibroin

Silk-based medical products have a long history of use as a material for surgical sutures because of their desirable mechanical properties. However, silk fibroin fabric has been reported to be haemolytic when in direct contact with blood. The layer-by-layer self-assembly technique provides a method for surface modification to improve the biocompatibility of silk fibroin fabrics. Regenerated silk fibroin and alginate, which have excellent biocompatibility and low immunogenicity, are outstanding candidates for polyelectrolyte deposition. In this study, silk fabric was degummed and positively charged to create a silk fibroin fabric that could undergo self-assembly. The multilayer self-assembly of the silk fibroin fabric was achieved by alternating the polyelectrolyte deposition of a negatively charged alginate solution (pH = 8) and a positively charged regenerated silk fibroin solution (pH = 2). Finally, the negatively charged regenerated silk fibroin solution (pH = 8) was used to assemble the outermost layer of the fabric so that the surface would be negatively charged. A stable structural transition was induced using 75% ethanol. The thickness and morphology were characterised using atomic force microscopy. The properties of the self-assembled silk fibroin fabric, such as the bursting strength, thermal stability and flushing stability, indicated that the fabric was stable. In addition, the cytocompatibility and haemocompatibility of the self-assembled silk fibroin fabrics were evaluated. The results indicated that the biocompatibility of the self-assembled multilayers was acceptable and that it improved markedly. In particular, after the self-assembly, the fabric was able to prevent platelet adhesion. Furthermore, other non-haemolytic biomaterials can be created through self-assembly of more than 1.5 bilayers, and we propose that self-assembled silk fibroin fabric may be an attractive candidate for anticoagulation applications and for promoting endothelial cell adhesion for vascular prostheses.     

绞股蓝皂苷-硒配合物对酪氨酸酶的活性影响及动力学分析

为考察绞股蓝皂苷及其硒配合物对酪氨酸酶的动力学参数和作用机理。本研究采用体外酶促反应,以L-酪氨酸和L-DOPA为底物,模拟了酪氨酸酶单酚和二酚酶的体外催化氧化过程。绞股蓝总皂苷在50%、70%乙醇洗脱段和50%、70%乙醇洗脱绞股蓝皂苷-硒配合物在酪氨酸酶上的Ki值分别为1. 533、1. 767、1. 312和1. 210 mmol/L。Ki值越低,对酪氨酸酶的抑制作用越强,单酚酶的氧化阶段越快,表明硒元素显著提高了绞股蓝皂苷对酪氨酸酶的抑制作用。酶反应动力学分析表明,四种绞股蓝皂苷及其硒配合物对酪氨酸的抑制作用均为混合竞争抑制。其独特的药理化学特性为绞股蓝及硒系美白化妆品的进一步研究开发提供了理论依据和参考。     

The great DOPA mystery: the source and significance of DOPA in phase I melanogenesis.

One of the important characteristics of tyrosinase is the autocatalytic nature of the oxidation of natural monohydric phenol substrates, such as tyrosine. In vitro tyrosinase exhibits a lag phase in which the maximum velocity of oxidation is attained after a period of induction. This acceleration contrasts with the kinetics of dihydric phenol oxidation which exhibit conventional Michaelis-Menten kinetics. It has been known for half a century that DOPA is a co-factor in the oxidation of tyrosine and addition of a small amount of catechol reduces the length of the lag period. The significance of DOPA is in this action, and DOPA is known to be formed in phase I melanogenesis. Until recently there has been controversy regarding the source of the DOPA in the in vitro reaction system. Most investigators have favoured a mechanism based on the generation of DOPA by a direct hydroxylation of tyrosine. However, recent evidence has suggested that DOPA is indirectly derived by reduction of dopaquinone. In this communication the evidence for the indirect mechanism derived from the use of analogue substrates is reviewed.     

Hydrothermal production and characterization of protein and amino acids from silk waste

Non-catalytic hydrothermal decomposition of sericin and fibroin from silk waste into useful protein and amino acids was examined in a closed batch reactor at various temperatures, reaction times, and silk to water ratios to examine their effects on protein and amino acid yields. For the decomposition of sericin, the highest protein yield was found to be 0.466 mg protein/mg raw silk, obtained after 10 min hydrothermal reaction of silk waste at 1:100 silk to water ratio at 120 degrees C. The highest amino acid yield was found to be 0.203 mg amino acids/mg raw silk, obtained after 60 min of hydrothermal reaction of silk waste at 1:20 silk to water ratio at 160 degrees C. For the hydrothermal decomposition of fibroin, the highest protein yield was 0.455 mg protein/mg silk fibroin (1:100, 220 degrees C, 10 min) and that of amino acids was 0.755 mg amino acids/mg silk fibroin (1:50, 220 degrees C, 60 min). The rate of silk fibroin decomposition could be described by surface reaction kinetics. The soluble reaction products were freeze-dried to obtain sericin and fibroin particles, whose conformation and crystal structure of the particles were shown to differ from the original silk materials, particularly in the case of fibroin, in which the change from beta-sheet conformation to alpha-helix/random coil was observed.     

Oxidation of peptides and proteins by Trichoderma reesei and Agaricus bisporus tyrosinases

The capability of a novel tyrosinase from Trichoderma reesei (TrTyr) to catalyse the oxidation and oxidative cross-linking of l-tyrosine (l-Y) and tyrosine side-chains in GYG and EGVYVHPV peptides, in bovine serum albumin (BSA) and beta-casein proteins as well as in proteinaceous wool fibres was studied by oxygen consumption measurement, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), reverse phase high-performance liquid chromatography (RP-HPLC), matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence microscopy. TrTyr was compared to the well-characterised tyrosinase from Agaricus bisporus (AbTyr) in terms of oxidation and cross-linking. According to the results obtained TrTyr was capable of cross-linking peptides and proteins more efficiently than AbTyr. However, the size and three-dimensional structure of the proteinaceous substrates proved to be crucial for the success of the enzymatic catalysis. Random coil beta-casein could be cross-linked by TrTyr already in three hours, but large and compact BSA was not cross-linked even in 24h. TrTyr could also be used to incorporate a diphenolic compound, l-dihydroxyphenyl alanine (l-dopa), into protein fibres whereas incorporation of a monophenol, l-Y was less efficient. Thus TrTyr is a potential tool for protein cross-linking and/or modification.     

Predominant synthesis of fibroin heavy and light chains on the membrane-bound polysomes prepared from the posterior silk gland of the silkworm, Bombyx mori

Membrane-bound polysomes were prepared from the posterior silk gland of the silkworm, Bombyx mori, on the fourth to fifth day in the fifth larval instar. The polysomes, when supplemented with a soluble fraction from the posterior silk gland, exhibited the elongation reaction of the growing polypeptide-chains, but the initiation reaction of polypeptide synthesis was not demonstrated in this system. The predominant products synthesized on the membrane-bound polysomes were fibroin heavy chain (H-chain) and light chain (L-chain), while polypeptides of heterogeneous size classes were synthesized on the 105,000 X g-sedimentable polysomes. A substantial fraction of the fibroin L-chain synthesized was bound to the H-chain by disulfide bond. Most of the newly synthesized fibroin H- and L-chains on the membrane-bound polysomes were proved to be present within microsomal membrane vesicles because of their insensitivity to digestion with proteases in the absence of Triton X-100.     

蜘蛛丝的分子结构与力学性能研究

蜘蛛丝尤其是蜘蛛大囊状腺产生的拖丝,具有独特的机械性能,是自然界颇具应用潜力的生物材料。现代分子生物学技术使蜘蛛丝蛋白基因得以克隆,通过高分子物理化学手段方法的利用,有利于揭示蜘蛛丝蛋白质序列、分子结构、以及分子结构和力学性能之间的关系。对不同种类蜘蛛丝蛋白的深入研究,将为基因工程方法人工合成并改造蜘蛛丝成为可能。