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1.
Using atomic force microscopy (AFM), the formation of nanosized silica structures on a substrate, catalyzed by the recombinant silicatein LoSilA1 from the marine sponge Latrunculia oparinae, was studied. It has been shown that at room temperature under neutral conditions, recombinant silicatein immobilized on a mica substrate causes the rapid polycondensation of tetrakis(2-hydroxyethyl) orthosilicate to form spherical particles. Thus, immobilized silicatein may acts as a catalyst in the preparation of ordered silica structures on various surfaces.  相似文献   

2.
Silicatein from Suberites domuncula was known to catalyze silica deposition in vitro under near neutral pH and ambient temperature conditions. In this study, we employed GST–glutathione (GSH) interaction system to increase the production of silicatein and develop an efficient protein immobilization method. Recombinant silicatein fused with GST (GST-SIL) was produced in E. coli and the GST-SIL protein was employed on GSH-coated glass plate. GST-SIL bound surface or matrix can catalyze the formation of silica layer in the presence of tetraethyl orthosilicate as a substrate at an ambient temperature and neutral pH. During silicatein-mediated silicification, green fluorescent protein (GFP) or horseradish peroxidase (HRP) can be efficiently immobilized on the silica surface. Immobilized GFP or HRP retained their activity and were released gradually. This biocompatible silica coating technique can be employed to prepare biomolecule-immobilized surfaces or matrixes, which are useful for the development of biocatalytic, diagnostic and biosensing system, or tissue culture scaffolds.  相似文献   

3.
A series of poly(dimethyl siloxane) (PDMS)/silica nanocomposites were synthesized utilizing a sol gel method. The samples were evaluated using pseudobarnacle adhesion and tensile strength tests. The effects of the molecular weight of the PDMS and the size and structure of the silica domains on biofouling release and the mechanical behavior of the PDMS/silica materials were investigated. Three different molecular weights (18,000, 49,000 and 79,000 g mol(-1)) of hydroxyl-terminated PDMS (HT-PDMS) were used to prepare the nanocomposites with three different weight ratios (1:1, 3:1 and 5:1) of HT-PDMS to tetraethyl orthosilicate (TEOS). TEOS served as a crosslinker to form PDMS networks and as a precursor to form silica domains. Two different variants of TEOS with regard to its degree of polymerization (n) (monomeric type: n ≈= 1 and oligomeric type: n ≈= 5) were used for in situ formation of silica particles via the sol-gel process. The mechanical properties of the composites were characterized using stress-strain isotherms. All the mechanical properties evaluated (Young's modulus, tensile strength, energy required for rupture, elongation at break) improved with increases in the molecular weight of the HT-PDMS and the silica content. The pseudobarnacle adhesion test was used to examine the fouling- release (FR) properties of coatings applied on aluminum plates. The rupture energy and tensile strength increased substantially when oligomeric TEOS was employed in the PDMS/silica composites. Scanning electron microscopy (SEM) was used to investigate the structure of the silica domains. It was found that the use of oligomeric TEOS in higher molecular weight PDMS samples with higher PDMS/TEOS weight ratios led to low pseudobarnacle adhesion strengths of ≈ 0.3 MPa, which is in the range of commercial FR coatings.  相似文献   

4.
《Process Biochemistry》2014,49(1):95-101
Silicatein has high sequence identity and similarity with that of cathepsin L. In silicatein, serine replaces the active-site cysteine that is found in cathepsin L. Here, we obtained hypothetical cathepsin-like protein (CAT) from Nematostella vectensis which is 55% identical and 75% similar to mature silicatein alpha (SIL) of Suberites domuncula. When this protein was expressed in Escherichia coli, it displayed protease activity with both N-carbobenzoxy-l-phenylalanyl-l-arginine-4-methylcoumaryl-7-amide (Z-FR-AMC) and gelatin substrates, as well as silica-condensing activity using the tetraethoxy silane (TEOS) substrate. To increase its silica-forming activity and stability, some residues including the active site cysteine, were mutated into conserved silicatein residues, resulting in a mutant with 65% identity and 79% similarity to SIL. The mutant silicatein-like cathepsin (SLC) had increased expression levels in E. coli, and silica-forming activity comparable to that of SIL. In addition, SLC exhibited decreased protease activity as compared to that of CAT. Both CAT and SLC produced silica particles of sizes smaller than 50 nm, which increased to 200–300 nm in the presence of a structure-directing agent, such as Triton X-100. In conclusion, CAT was evolved to function as a biosilica-forming protein, and SLC was engineered by mutating CAT residues into conserved SIL residues to produce various silica-based materials.  相似文献   

5.
Silicateins are unique enzymes of sponges (phylum Porifera) that template and catalyze the polymerization of nanoscale silicate to siliceous skeletal elements. These multifunctional spicules are often elaborately shaped, with complex symmetries. They carry an axial proteinaceous filament, consisting of silicatein and the scaffold protein silintaphin-1, which guides silica deposition and subsequent spicular morphogenesis. In vivo, the synthesis of the axial filament very likely proceeds in three steps: (a) assembly of silicatein monomers to form one pentamer; (b) assembly of pentamers to form fractal-like structures; and finally (c) assembly of fractal-like structures to form filaments. The present study was aimed at exploring the effect of self-assembled complexes of silicatein and silintaphin-1 on biosilica synthesis in vitro. Hence, in a comparative approach, recombinant silicatein and recombinant silintaphin-1 were used at different stoichiometric ratios to form axial filaments and to synthesize biosilica. Whereas recombinant silicatein-α reaggregates to randomly organized structures, coincubation of silicatein-α and silintaphin-1 (molecular ratio 4 : 1) resulted in synthetic filaments via fractal-like patterned self-assemblies, as observed by electron microscopy. Concurrently, owing to the concerted action of both proteins, the enzymatic activity of silicatein-α strongly increased by 5.3-fold (with the substrate tetraethyl orthosilicate), leading to significantly enhanced synthesis of biosilica. These results indicate that silicatein-α-mediated biosilicification depends on the concomitant presence of silicatein-α and silintaphin-1. Accordingly, silintaphin-1 might not only enhance the enzymatic activity of silicatein-α, but also accelerate the nonenzymatic polycondensation of the silica product before releasing the fully synthesized biosiliceous polymer.  相似文献   

6.
Siliceous sponges can synthesize poly(silicate) for their spicules enzymatically using silicatein. We found that silicatein exists in silica-filled cell organelles (silicasomes) that transport the enzyme to the spicules. We show for the first time that recombinant silicatein acts as a silica polymerase and also as a silica esterase. The enzymatic polymerization/polycondensation of silicic acid follows a distinct course. In addition, we show that silicatein cleaves the ester-like bond in bis(p-aminophenoxy)-dimethylsilane. Enzymatic parameters for silica esterase activity are given. The reaction is completely blocked by sodium hexafluorosilicate and E-64. We consider that the dual function of silicatein (silica polymerase and silica esterase) will be useful for the rational synthesis of structured new silica biomaterials.  相似文献   

7.
Bioprinting/3D cell printing procedures for the preparation of scaffolds/implants have the potential to revolutionize regenerative medicine. Besides biocompatibility and biodegradability, the hardness of the scaffold material is of critical importance to allow sufficient mechanical protection and, to the same extent, allow migration, cell–cell, and cell–substrate contact formation of the matrix‐embedded cells. In the present study, we present a strategy to encase a bioprinted, cell‐containing, and soft scaffold with an electrospun mat. The electrospun poly(?‐caprolactone) (PCL) nanofibers mats, containing tetraethyl orthosilicate (TEOS), were subsequently incubated with silicatein. Silicatein synthesizes polymeric biosilica by polycondensation of ortho‐silicate that is formed from prehydrolyzed TEOS. Biosilica provides a morphogenetically active matrix for the growth and mineralization of osteoblast‐related SaOS‐2 cells in vitro. Analysis of the microstructure of the 300–700 nm thick PCL/TEOS nanofibers, incubated with silicatein and prehydrolyzed TEOS, displayed biosilica deposits on the mats formed by the nanofibers. We conclude and propose that electrospun PCL nanofibers mats, coated with biosilica, may represent a morphogenetically active and protective cover for bioprinted cell/tissue‐like units with a suitable mechanical stability, even if the cells are embedded in a softer matrix.  相似文献   

8.
9.
The siliceous spicules of sponges (Porifera) are synthesized by the enzyme silicatein. This protein and its gene have been identified so far in the Demospongiae, e.g., Tethya aurantium and Suberites domuncula. In the Hexactinellida, the second class of siliceous sponges, the mechanism of synthesis of the largest bio-silica structures on Earth remains obscure. Here, we describe the morphology of the spicules (diactines and stauractines) of the hexactinellid Crateromorpha meyeri. These spicules are composed of silica lamellae concentrically arranged around a central axial canal and contain proteinaceous sheaths (within the siliceous mantel) and proteinaceous axial filaments (within the axial canal). The major protein in the spicules is a 24-kDa protein that strongly reacts with anti-silicatein antibodies in Western blots. Its cDNA has been successfully cloned; the deduced hexactinellid silicatein comprises, in addition to the characteristic catalytic triad amino acids Ser-His-Asn and the "conventional" serine cluster, a "hexactinellid C. meyeri-specific" Ser cluster. We show that anti-silicatein antibodies react specifically with the proteinaceous matrix of the C. meyeri spicules. The characterization of silicatein at the genetic level should contribute to an understanding of the molecular/biochemical mechanism of spiculogenesis in Hexactinellida. These data also indicate that silicatein is an autapomorphic molecule common to both classes of siliceous sponges.  相似文献   

10.
In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.  相似文献   

11.
We examined the performance of methyltrimethoxysilane (MTMS), a precursor of silicic acid, in the process of biosilicification induced by the R5 peptide from Cylindrotheca fusiformis. Recombinant GFP-R5 fusion protein was produced by Escherichia coli cultured at 25°C as a soluble and functional formation, but not at 37°C. MTMS-based biosilica deposits had a larger average diameter compared to tetraethyl orthosilicate (TEOS)-based deposits. Reducing phosphate concentration in the buffer system led to a decrease in the size of MTMS-based biosilica. These results provide insight into the surface modification of biosilica, and control of biosilica particle size, when using hydrophobic precursors such as MTMS.  相似文献   

12.
Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan δ solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials'' morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by Stöber''s methods, which have smooth surfaces.  相似文献   

13.
Recently it has been discovered that the formation of the siliceous spicules of Demospongiae proceeds enzymatically (via silicatein) and occurs matrix guided (on galectin strings). In addition, it could be demonstrated that silicatein, if immobilized onto inorganic surfaces, provides the template for the synthesis of biosilica. In order to understand the formation of spicules in the intact organism, detailed studies with primmorphs from Suberites domuncula have been performed. The demosponge spicules are formed from several silica lamellae which are concentrically arranged around the axial canal, harboring the axial filament composed of silicatein. Now we show that the appositional growth of the spicules in radial and longitudinal direction proceeds in the extracellular space along hollow cylinders; their surfaces are formed by silicatein. The extracellularly located spicules are surrounded by sclerocytes which are filled with both electron-dense and electron-poor vesicles; energy dispersive X-ray analysis/scanning electron microscopical studies revealed that the electron-dense vesicles are filled of silicon/silica and therefore termed silicasomes. The release of the content of the silicasomes into the hollow cylinder suggests that the newly formed silica lamella originate there; in addition the data are compatible with the view that the silicatein molecules, attached at the centripetal and centrifugal surfaces, mediate biosilica formation. In a chemical/biomimetical approach silicatein is linked onto the organic material-free spicules after their functionalization with aminopropyltriethoxysilane [amino groups]-poly(acetoxime methacrylate) [reactive ester polymer]-N(epsilon)-benzyloxycarbonyl L-lysine tert-butyl ester-Ni(II); finally His-tagged silicatein is immobilized. The matrix-bound enzyme synthesized a new biosilica lamella. These bioinspired findings are considered as the basis for a technical use/application/utilization of hollow cylinders formed by matrix-guided silicatein molecules for the biocatalytic synthesis of nanostructured tubes.  相似文献   

14.
While most forms of multicellular life have developed a calcium-based skeleton, a few specialized organisms complement their body plan with silica. However, of all recent animals, only sponges (phylum Porifera) are able to polymerize silica enzymatically mediated in order to generate massive siliceous skeletal elements (spicules) during a unique reaction, at ambient temperature and pressure. During this biomineralization process (i.e., biosilicification) hydrated, amorphous silica is deposited within highly specialized sponge cells, ultimately resulting in structures that range in size from micrometers to meters. Spicules lend structural stability to the sponge body, deter predators, and transmit light similar to optic fibers. This peculiar phenomenon has been comprehensively studied in recent years and in several approaches, the molecular background was explored to create tools that might be employed for novel bioinspired biotechnological and biomedical applications. Thus, it was discovered that spiculogenesis is mediated by the enzyme silicatein and starts intracellularly. The resulting silica nanoparticles fuse and subsequently form concentric lamellar layers around a central protein filament, consisting of silicatein and the scaffold protein silintaphin-1. Once the growing spicule is extruded into the extracellular space, it obtains final size and shape. Again, this process is mediated by silicatein and silintaphin-1, in combination with other molecules such as galectin and collagen. The molecular toolbox generated so far allows the fabrication of novel micro- and nanostructured composites, contributing to the economical and sustainable synthesis of biomaterials with unique characteristics. In this context, first bioinspired approaches implement recombinant silicatein and silintaphin-1 for applications in the field of biomedicine (biosilica-mediated regeneration of tooth and bone defects) or micro-optics (in vitro synthesis of light waveguides) with promising results.  相似文献   

15.
Mixed titania/silica xerogels were prepared using titanium tetraisopropoxide (TTIP) and tetraethoxy orthosilicate (TEOS). Xerogel properties were modified by incorporating n-octyltriethoxysilane (C8). The xerogels catalyze the oxidation of bromide and chloride with hydrogen peroxide (H2O2) to produce hypohalous acids at pH 7 and pH 8. The antifouling/ fouling-release performance of a TTIP/C8/TEOS xerogel in the presence and absence of H2O2 was evaluated for the settlement of zoospores of the marine alga Ulva linza and for the removal of sporelings (young plants). In the absence of H2O2, differences in the settlement of zoospores and removal of sporelings were not significant relative to a titanium-free C8/TEOS xerogel. Addition of H2O2 gave a significant reduction in zoospore settlement and sporeling removal relative to the C8/TEOS xerogel and relative to peroxide-free conditions. The impact of TTIP on xerogel characteristics was evaluated by comprehensive contact angle analysis, scanning electron microscopy, and X-ray photoelectron spectroscopy.  相似文献   

16.
The glass sponge Monorhaphis chuni (Porifera: Hexactinellida) forms the largest bio-silica structures on Earth; their giant basal spicules reach sizes of up to 3 m and diameters of 8.5 mm. Previously, it had been shown that the thickness growth proceeds by appositional layering of individual lamellae; however, the mechanism for the longitudinal growth remained unstudied. Now we show, that the surface of the spicules have towards the tip serrated relief structures that are consistent in size and form with the protrusions on the surface of the spicules. These protrusions fit into the collagen net that surrounds the spicules. The widths of the individual lamellae do not show a pronounced size tendency. The apical elongation of the spicule proceeds by piling up cone-like structural units formed from silica. As a support of the assumption that in the extracellular space silicatein(-like) molecules exist that associate with the external surface of the respective spicule immunogold electron microscopic analyses were performed. With the primmorph system from Suberites domuncula we show that silicatein(-like) molecules assemble as string- and net-like arrangements around the spicules. At their tips the silicatein(-like) molecules are initially stacked and at a later stay also organized into net-like structures. Silicatein(-like) molecules have been extracted from the giant basal spicule of Monorhaphis. Applying the SDS–PAGE technique it could be shown that silicatein molecules associate to dimers and trimers. Higher complexes (filaments) are formed from silicatein(-like) molecules, as can be visualized by electron microscopy (SEM). In the presence of ortho-silicate these filaments become covered with 30–60 nm long small rod-like/cuboid particles of silica. From these data we conclude that the apical elongation of the spicules of Monorhaphis proceeds by piling up cone-like silica structural units, whose synthesis is mediated by silicatein(-like) molecules.  相似文献   

17.
Tetrakis(2-hydroxyethyl) orthosilicate (THEOS) introduced by Hoffmann et al. (J. Phys. Chem. B., 106 (2002) 1528) was first used to prepare hybrid nanocomposites containing various polysaccharides and immobilize enzymes in these materials. Two different types of O-glycoside hydrolyses (EC3.2.1), 1-->3-beta-D-glucanase LIV from marine mollusk Spisula sacchalinensis and alpha-D-galactosidase from marine bacterium Pseudoalteromonas sp. KMM 701, were taken for the immobilization. To reveal whether the polysaccharide inside the hybrid material influences the enzyme entrapment and functioning, negatively charged xanthan, cationic derivative of hydroxyethylcellulose and uncharged locust bean gum were examined. The mechanical properties of these nanocomposites were characterized by a dynamic rheology and their structure by a scanning electron microscopy. It was found that 1-->3-beta-D-glucanase was usually immobilized without the loss of its activity, while the alpha-D-galactosidase activity in the immobilized state depended on the polysaccharide type of material. An important point is that the amount of immobilized enzymes was small, comparable to their content in the living cells. It was shown by the scanning electron microscopy that the hybrid nanocomposites are sufficiently porous that allows the enzymatic substrates and products to diffuse from an external aqueous solution to the enzymes, whereas protein molecules were immobilized firmly and not easily washed out of the silica matrix. A sharp increase of the enzyme lifetime (more than a hundred times) was observed after the immobilization. As established, the efficient entrapment of enzymes is caused by few advantages of new precursor over the currently used TEOS and TMOS: (i) organic solvents and catalysts are not needed owing to the complete solubility of THEOS in water and the catalytic effect of polysaccharides on the sol-gel processes; (ii) the entrapment of enzymes can be performed at any pH which is suitable for their structural integrity and functionality; (iii) a gel can be prepared at reduced concentrations of THEOS (1-2%) in the initial solution that excludes a notable heat release in the course of its hydrolysis.  相似文献   

18.
Biogenetic silica displays intricate patterns assembling from nano- to microsize level and interesting non-spherical structures differentiating in specific directions. Several model systems have been proposed to explain the formation of biosilica nanostructures. Of them, phase separation based on the physicochemical properties of organic amines was considered to be responsible for the pattern formation of biosilica. In this paper, using tetraethyl orthosilicate (TEOS, Si(OCH2CH3)4) as silica precursor, phospholipid (PL) and dodecylamine (DA) were introduced to initiate phase separation of organic components and influence silica precipitation. Morphology, structure and composition of the mineralized products were characterized using a range of techniques including field emission scanning electron microscopy (FESEM), transmission electron microscope (TEM), X-ray diffraction (XRD), thermogravimetric and differential thermal analysis (TG-DTA), infrared spectra (IR), and nitrogen physisorption. The results demonstrate that the phase separation process of the organic components leads to the formation of asymmetrically non-spherical silica structures, and the aspect ratios of the asymmetrical structures can be well controlled by varying the concentration of PL and DA. On the basis of the time-dependent experiments, a tentative mechanism is also proposed to illustrate the asymmetrical morphogenesis. Therefore, our results imply that in addition to explaining the hierarchical porous nanopatterning of biosilica, the phase separation process may also be responsible for the growth differentiation of siliceous structures in specific directions. Because organic amine (e.g., long-chair polyamines), phospholipids (e.g., silicalemma) and the phase separation process are associated with the biosilicification of diatoms, our results may provide a new insight into the mechanism of biosilicification.  相似文献   

19.
Andre R  Tahir MN  Natalio F  Tremel W 《The FEBS journal》2012,279(10):1737-1749
Owing to their physical and chemical properties, inorganic functional materials have tremendous impacts on key technologies such as energy generation and storage, information, medicine, and automotive engineering. Nature, on the other hand, provides evolution-optimized processes, which lead to multifunctional inorganic-bio-organic materials with complex structures. Their formation occurs under physiological conditions, and is goverened by a combination of highly regulated biological processes and intrinsic chemical properties. Nevertheless, insights into the molecular mechanisms of biomineralization open up promising perspectives for bioinspired and biomimetic design and the development of inorganic-bio-organic multifunctional hybrids. Therefore, biomimetic approaches may disclose new synthetic routes under ambient conditions by integrating the concept of gene-regulated biomineralization principles. The skeletal structures of marine sponges provide an interesting example of biosilicification via enzymatically controlled and gene-regulated silica metabolism. Spicule formation is initiated intracellularly by a fine-tuned genetic mechanism, which involves silica deposition in vesicles (silicassomes) under the control of the enzyme silicatein, which has both catalytic and templating functions. In this review, we place an emphasis on the fabrication of biologically inspired materials with silicatein as a biocatalyst.  相似文献   

20.
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