WO2005106003A1 - Enzym- und template-gesteuerte synthese von silica aus nicht-organischen siliciumverbindungen sowie aminosilanen und silazanen und verwendung - Google Patents
Enzym- und template-gesteuerte synthese von silica aus nicht-organischen siliciumverbindungen sowie aminosilanen und silazanen und verwendung Download PDFInfo
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- Silicon compounds are of exceptional economic importance. You will u. a. used in the glass, fiberglass and porcelain industry, in the cement production, for the production of ceramics, in the paint, rubber, plastic and paper industry, in the detergent industry, in the paint, Soap and cosmetics production as well as in medicine / dentistry, e.g. B. at the dental manufactory / repair. Certain silicates have molecular sieve and ion exchange properties as well as catalytic properties (see, inter alia: CD Römpp Chemie Lexikon - Version 1.0, Stuttgart / New York: Georg Thieme Verlag 1995).
- the end product of the condensation is a polymeric silicon dioxide (Si0 2 ) x , which is amorphous because chain-lengthening and branching processes take place in a disorderly manner during the condensation.
- Si0 2 ) x polymeric silicon dioxide
- the silicon atoms are at the center of regular tetrahedra, the corners of which form four oxygen atoms.
- the oxygen atoms simultaneously belong to the neighboring, irregularly linked tetrahedra.
- the skeleton of the diatoms (diatoms) and the sponges consists of amorphous Si0 2 ("Biosilica").
- the Si0 2 synthesis in these organisms is characterized by high (structural) specificity and controllability, which enables the synthesis of defined structures in the microscopic and submicroscopic range (nanostructures).
- silica sponges have the ability to form their silicate frameworks under mild conditions, ie at a relatively low temperature and pressure. This is due to the fact that specific enzymes are involved in their synthesis. In contrast, drastic conditions such as high pressure and high temperature are usually required for the chemical synthesis of the silicates. That is why the production of many silicon compounds using conventional methods is cost-intensive and also not very environmentally friendly.
- the first enzyme is silicatein, which occurs in three forms, silicatein- ⁇ , -ß and - ⁇ (PCT / US99 / 30601.
- Methods, compositions, and biomimetic catalysts, such as silicateins and block copolypeptides used to catalyze and spatially direct the polycondensation of Silicon alkoxides, metal alkoxides, and their organic conjugates to make silica, polysiloxanes, polymetallo-oxanes, and mixed poly (silicon / met-allo) oxane materials under environmentally benign conditions.
- Silicatein- ⁇ was cloned from the marine pebble sponge Suberites domuncula (Krasko A, Batel R, Schröder HC, Müller IM, Müller WEG (2000) Expression of silicatein and collagen genes in the marine sponge S. domuncula is controlled by silicate and myotrophin. Europ J Biochem 267: 4878-4887).
- Silicatein-ß which was also cloned from S. domuncula, has several advantageous properties compared to silicatein- ⁇ in terms of its catalytic abilities and their technical / medical applicability. (DE 103 52 433.9. Enzymatic synthesis, modification and degradation of silicon (IV) and other metal (IV) compounds. German Patent Office 2003. Applicant: Johannes Gutenberg University Mainz; inventor: Müller WEG, Schwertner H , Schröder HC).
- silicateine, silicatein- ⁇ and silicatein- ⁇ are only able to form amorphous silicon dioxide (polysilicic acids and polysilicates) from organic silicon compounds (alkoxysilanes) (Cha JN, Shimizu K, Zhou Y, Christianssen SC , Chmelka BF, Stucky GD, Morse DE (1999) Silicatein filaments and subunits from a marine sponge direct the polymerization of silica and silicones in vitro. Proc Natl Acad Sei USA 96: 361-365; and patents cited above).
- the second enzyme is a silicase (DE 102 46 186.4. Degradation and modification of silicates and silicones by silicase and use of the reversible enzyme. German Patent Office 2002. Applicant: Johannes Gutenberg University Mainz; inventor: Müller WEG, Krasko A , Schröder HC; PCT / EP03 / 10983. Degradation and modification of silicates and silicones by silicase and use of the reversible enzyme. European Patent Office 2003. Applicant: Johannes Gutenberg University Mainz.
- Silicase an enzyme which degrades biogenous amorphous silica: Contribution to the metabolism of silica deposition in the demosponge Suberites domuncula Prog Mol Subcell Biol 33: 250-268).
- Silicase in particular the enzyme from the S. domuncula sea sponge, is able to dissolve both amorphous and crystalline silicon dioxide. This releases silica.
- the silicase has the ability to dissolve lime material in analogy to the carbonic anhydrase.
- this enzyme in the presence of a suitable template (e.g. collagen), is also capable of synthesizing amorphous silicon dioxide (silica) from non-organic short-chain metasilicates and from one or more Si-N bonds containing aminosilanes or silazanes.
- a suitable template e.g. collagen
- collagen is a main component of the extracellular matrix of tissues and organs.
- Collagen fibrils have an extremely high tensile strength. As a result, they are particularly able to give the connective and support tissue mechanical stability. The formation of collagen fibrils is also an important process in wound healing.
- the collagens form a large family of proteins: 19 types of collagen have been described which are encoded by at least 33 different genes (Prockop and Kivirikko (1995) Annu Rev Biochem 64: 403-434).
- Members of the collagen family include both fibrillary and non-fibrillar proteins.
- Fibrillar collagen types I, II, III, V and XI are able to form fibrils with a band pattern.
- the so-called non-fibrillary collagens occur together with the fibrillary collagens (fibril-associated collagens) or in the basement membranes (type IV; basement membrane collagens). This group also includes short-chain collagens.
- Some collagens - like types XV and XVIII - are only known because of their cDNA.
- the common structural feature of all collagens is the triple helix, which consists of three twisted polypeptide chains ( ⁇ chains), which have the repeating sequence G-x-y; x is mostly proline and y is often hydroxyproline.
- This triplet determines the characteristic helical conformation of the collagen ⁇ -helix and its property to assemble with similar polypeptide chains to form the triple helix (Brodsky and Ramshaw (1997) Matrix Biol 15: 545-554).
- the triple helix is usually composed of the polypeptide chains of different collagen types ( ⁇ 1, ⁇ 2, ⁇ 3).
- the resulting structure is highly stable due to the position of glycine (a small amino acid) close to the axis of the helix, the stabilizing effect of proline and the formation of hydrogen bonds (Bella et al. (1994) Science 266: 75-81).
- Type I collagen forms the main amount of collagen in the organism.
- Type II collagen is the fibril-forming collagen of the cartilage. at In each of these collagen types there are three ⁇ chains. The length of the tropocollagen molecules thus formed is 280 nm. An offset arrangement of these building blocks is found in the collagen fibrils. The staggered arrangement of these molecules causes transverse streaks to appear within the collagen fibers every 68 nm.
- Type IV collagen is specialized for the formation of spatial lattices and occurs in the basement membranes.
- Type VI collagen found in the interstitial connective tissue has only a relatively short triple helix; the two globular domains at the ends of this dumbbell-type collagen interact with type I collagen and membrane-bound integrins.
- the type VIII collagen is used to anchor the basement membrane under squamous epithelia.
- the Type VIII and Type X collagens are short chain collagens; the type VIII collagens associate into a hexagonal network.
- Type IX collagen belongs to the fibril-associated collagens and occurs together with type I I collagen in the calcifying areas of the enchondral cartilage.
- Collagen is also a major protein in the sponges' extracellular matrix and acts as a matrix for spicule formation (sponge needle formation) (Krasko et al. (2000) Eur J Biochem 267: 4878-4887). Collagen fibrils in sponges are very similar to those in vertebrates (Gross et al. (1956) J Histochem Cytochem 4: 227-246; Garrone et al. (1975) J Ultrastruct Res 52: 261-275; Garrone (1978) Physiogenesis of connective tissue. Karger, Basel).
- FASEB J 14: 2022-2031 consists of (i) a non-collagenic ⁇ / -terminal domain, (ii) a collagenous internal domain and (iii) a non-collagenic C-terminal domain ,
- the internal domain in S. domuncula is unusually short with only 24 Gxy collagen triplets.
- the collagen of the fresh water sponge Ephydatia muelleri has two internal domains with 79 Gxy triplets (Exposito et al. (1991) J Biol Chem 266: 21923-21928).
- the organization of the genes coding for the fibrillar sponge collagens is thus very similar to that of the vertebrate collagen genes.
- silicase and other carbonic anhydrases and silicateins are capable, in the presence of suitable templates such as collagen, of also non-organic silicon compounds, in particular metasilicates, and aminosilanes containing one or more Si-N bonds or to implement silazanes in silica.
- suitable templates such as collagen, of also non-organic silicon compounds, in particular metasilicates, and aminosilanes containing one or more Si-N bonds or to implement silazanes in silica.
- silicateins catalyze the hydrolysis of organic silicon compounds with one or more Si-O bonds (alkoxysilanes) (with subsequent condensation of the released silanols to form amorphous silicon dioxide; see Zhou et al. (1999) Angew. Chem. [ Int.
- a method for the in vitro or in vivo synthesis of amorphous silicon dioxide (silica, condensation products of silicic acid) and other metal (IV) compounds is thus generally provided, using a polypeptide or a metal complex of a polypeptide which is either characterized in that the polypeptide comprises an animal, plant, bacterial or fungal carbonic anhydrase domain which has at least 25%, preferably at least 50%, more preferably at least 75% and most preferably at least 95% sequence identity to the in SEQ ID No.
- polypeptide comprises an animal, bacterial, plant or fungus silicatein- ⁇ -domain or silicatein- ⁇ -domain which is at least 25%, preferably at least 50%, further preferably at least 75 % and most preferably at least 95% sequence identity to that shown in SEQ ID No. 3 and SEQ ID No. 5, respectively sequence.
- Another aspect of the present invention relates to the use of a template which is a polypeptide of collagen from S. domuncula according to SEQ ID No. 7 or a polypeptide which is homologous thereto and which has at least 25%, preferably at least 50%, more preferably at least 75 in its amino acid sequence % and most preferably at least 95% has sequence identity to, or contains, or consists of, the sequence shown in SEQ ID No. 7.
- the template (collagen or other polypeptide) according to the invention can be characterized in that it has been produced synthetically or in that it is present in a prokaryotic or eukaryotic cell extract or lysate.
- the cell extract or lysate can be obtained from a cell ex vivo or ex vitro, for example a recombinant bacterial cell or a sea sponge.
- the template according to the invention (collagen or other polypeptide) can be purified by conventional methods known in the art and can therefore be essentially free of other proteins.
- a method according to the invention is preferred which is characterized in that compounds such as silicas (orthosilicic acid and metasilicic acid) or their salts (orthosilicates and metasilicates) or other metal (IV) compounds are used as reactants (substrates) for the synthesis.
- compounds such as silicas (orthosilicic acid and metasilicic acid) or their salts (orthosilicates and metasilicates) or other metal (IV) compounds are used as reactants (substrates) for the synthesis.
- a method according to the invention which is characterized in that compounds such as alkyl- and dialkylaminosilanes, bis (alkylamino) silanes or bis (dialkylamino) silanes, tris (alkylamino) silanes or tris (dialkylamino) silanes, tetrakis ( alkylamino) silanes or tetrakis (dialkylamino) silanes and alkyl- or aryl-substituted derivatives of these compounds (generally: aminosilanes), which are characterized in that they contain one or more Si-N bonds.
- compounds such as alkyl- and dialkylaminosilanes, bis (alkylamino) silanes or bis (dialkylamino) silanes, tris (alkylamino) silanes or tris (dialkylamino) silanes, tetrakis ( alkylamino) silanes or tetrakis (dialky
- a method according to the invention which is characterized in that di-, tri-, tetra- and polysilazanes as well as alkyl- or aryl-substituted derivatives of these compounds (generally: silazanes), including the cyclic compounds (cyclotrisilazanes, cyclotetrasilazanes and other derivatives).
- Another aspect of the present invention is the use of the method for modifying surfaces of glass, metals, metal oxides, plastics, biopolymers or other materials.
- the method can be used for the synthesis of defined two- and three-dimensional structures from amorphous silicon dioxide (silica, condensation products of silicic acid) and other polymeric metal (IV) compounds.
- amorphous silicon dioxide sica, condensation products of silicic acid
- IV polymeric metal
- a still further aspect of the present invention relates to a chemical compound or silica (amorphous silicon dioxide) -containing structure or surface which has been obtained by the method according to the invention.
- SEQ ID No. 2 shows the nucleotide sequence of the sponge silicase cDNA and SEQ ID No. 1 shows the polypeptide of the sponge silicase derived from the nucleotide sequence (SlA_SUBDO).
- the deduced amino acid sequence of the sponge silicase is very similar to the amino acid sequences of the carbonic anhydrase family.
- the eukaryote-type carbonic anhydrase domain PFAM00194
- the carbonic anhydrases form a family of zinc metal enzymes (Sly and Hu (1995) Annu Rev Biochem 64: 375-401).
- the three zinc-binding conserved histidine residues are found in the silicase at amino acids aa-is-i, aa-i83 and aa 206 (see SEQ ID No. 1).
- carbonic anhydrases from other organisms (some of which are commercially available) can also be used in the process according to the invention.
- SEQ ID No. 1 The amino acid sequence of the silicase from S. domuncula (SIA_SUBDO) used according to the invention.
- SEQ ID No. 2 The nucleic acid sequence of the silicase from S. domuncula used according to the invention.
- SEQ ID No. 3 The amino acid sequence of the silicate one- ⁇ from S. domuncula (SIA_SUBDO) used according to the invention.
- SEQ ID No. 4 The nucleic acid sequence of the silicate one- ⁇ from S. domuncula used according to the invention.
- SEQ ID No. 5 The amino acid sequence of the silicate one- ⁇ from S. domuncula (SIA_SUBDO) used according to the invention.
- SEQ ID No. 6 The nucleic acid sequence of the silicate one- ⁇ from S. domuncula used according to the invention.
- SEQ ID No. 7 The amino acid sequence of collagen 3 from S. domuncula (SIA_SUBDO) used according to the invention.
- SEQ ID No. 8 The nucleic acid sequence of collagen 3 from S. domuncula used according to the invention.
- Protein information about the proteins is: Protein information about CAexpresL.prt (long form): Molecular weight: 43130.74 Dalton + 25000 Da GST ⁇ > 68 kDa
- Proteins (long and short form of silicase). Protein information about the proteins is:
- Figure 4 Expression of non-fibrillar collagen 3 from S. domuncula in the pBAD / glll expression vector. From top to bottom are shown: nucleotide sequence of the collagen 3 clone with binding sites of the "forward primer” and the "reverse primer”; inserted sequence of the non-fibrillary collagen 3 of S.
- domuncula in the expression vector pBAD / glll (the restriction sites of ⁇ / col and H / ndlll are underlined); the primers used for expression in pBAD / glll ("forward primer” Col3_f and "reverse primer” Col 3_r; the restriction sites of ⁇ / col and / - / ' ndlll are marked); amino acid sequence of the recombinant protein derived from the nucleotide sequence.
- Sponge collagens A. Comparison of the deduced amino acid sequences of the cDNA of the S. domuncula collagen (COL1_SUBDO) with those of the collagen from E. muelleri (COL4_EPHMU). conserveed amino acid residues (similar or related in terms of their physicochemical properties) in the sequences are shown in white on black. NC1: non-collagenic ⁇ / -terminal domain. COL: collagen internal domain. NC2: non-collagenic C-terminal domain. B. Comparison of S. domuncu / a collagen with the collagen of E. muelleri. NC1: non-collagenic N-terminal domain. COL: collagen internal domain. NC2: non-collagenic C-terminal domain. Numbers: number of amino acids.
- the amount of SiO 2 formed increased sharply with an increasing amount of collagen (1.2 to 10 ⁇ g / ml) (from 0.022 to 0.023 to 0.068 to 0.070 OD units).
- An increase in the Na metasilicate concentration did not lead to any further increase, but rather to a decrease in the SiO 2 formation (up to 0.027 OD units).
- BSA bovine serum albumin
- very little Si0 2 was formed (0.008 OD units); however, in the presence of carbonic anhydrase alone, the Si0 2 formation was 0.019-0.029 OD units. Without the addition of catechol, the Si0 2 formation was somewhat lower.
- the concentrations given are the final concentrations after adding all components to the batches. To detect the amorphous silicon dioxide formed, the reaction batches were further treated as described in FIG. 5 and the amount of insoluble SiO 2 formed was determined.
- the concentrations given are the final concentrations after adding all components to the batches.
- the reaction batches were treated further as described in FIG. 5 and the amount of insoluble SiO 2 formed was determined.
- the results show that with the addition of increasing amounts of fibrillar collagen (cattle) - in contrast to recombinant, non-fibrillary sponge collagen - the amount of insoluble Si0 2 formed initially increases, but then drops again.
- the amount of SiO 2 formed increased sharply with increasing amount of sponge collagen (1 to 4 ⁇ g / ml) (from 0.002 to 0.010 OD units). Similar to the results obtained with Na metasilicate (see FIG.
- the evidence of the silica products formed is shown with the aid of a "High Performance Field Emission Electron Probe Microanaiyzer (EPMA)".
- the results of the element analysis for Si are shown in a batch with carbonic anhydrase and collagen (A) and a control (absence of carbonic anhydrase and collagen; B).
- the cDNA SDSIA) coding for the silicase from the sea sponge S. domuncula and the polypeptide (SIA_SUBDO) derived from the nucleotide sequence have the following properties. Length of the cDNA: 1395 nucleotides (nt); open reading grid: from nt ⁇ 22 - nt ⁇ 24 to nt ⁇ 259 - nt ⁇ 26 i (stop codon); Length of the polypeptide: 379 amino acids; molecular weight (M r ) of the polypeptide: 43131; isoelectric point (pl): 6.5.
- the recombinant S. domuncula silicase was produced as a glutathione S-transferase (GST) fusion protein.
- GST glutathione S-transferase
- Both a long and a shortened fragment of the cDNA coding for S. domuncula silicase (called: SDSIA) were cloned into a pGEX-4T-2 plasmid which contained the GST gene (FIG. 2).
- the results for the purified short form of the silicase with a size of 32 kDa are shown below; Analogous results are obtained for the long form (M r 43 kDa), which is however less efficient.
- the cDNA sequence (short form) coding for the silicase is amplified by PCR using the following primers: Forward primer: ATACTC GAG TCG AAA TGC CAC CGT CAC TTC TCC ACA TCA and Reverse primer: ATATCT AGA AA CCA ATA TAT CTT CCT GAC CAG CTC TCT; and cloned into pBAD / glllA (restriction nucleases for insertion in the expression onsvektor: Xnol and Xba ⁇ ). After transformation of E. coli XL1-Blue, the expression of the fusion protein with L-arabinose is induced.
- the recombinant silicatein- ⁇ was produced in E. coli using the oligo-histidine expression vector pBAD / glllA (Invitrogen), in which the recombinant protein is secreted into the periplasmic space on the basis of the gene III signal sequence .
- the cDNA sequence (short form) coding for the silicatein- ⁇ was amplified by PCR using the following primers: forward primer: TAT CC ATG GAC TAC CCT GAA GCT GTA GAC TGG AGA ACC and reverse primer: TAT T CTA GA A TTA TAG GGT GGG ATA AGA TGC ATC GGT AGC; and cloned into pBAD / glllA (restriction nucleases for insertion into the expression vector: ⁇ / col and Xba ⁇ ).
- the recombinant sponge silicatein polypeptide (short form) has a molecular weight of -28.5 kDa ( ⁇ 26 kDa silicatein plus 2 kDa vector) and an isol-electric point of pI 6.16.
- Both native collagen from vertebrates such as bovine collagen and from invertebrates (such as from marine demospongia) as well as recombinant collagen (in particular from the marine sponge S. domuncula) can be used as a template. Some methods for their presentation are described below ,
- the clone used to produce the recombinant collagen codes for a non-fibrillary collagen (collagen 3) from the sea sponge Suberites domuncula; this collagen has the advantage that it (1) has a relatively low molecular weight and (2) is not further modified post-translationally.
- the cDNA sequence coding for the sponge collagen 3 can be amplified by means of PCR using suitable primers and converted into a suitable expression vector to be subcloned.
- the expression was carried out successfully, inter alia, with the bacterial oligo-histidine expression vectors pBAD / glllA (Invitrogen) and pQTK_1 (Qiagen).
- the expression vector pBAD / glllA has the advantage that, based on the gene III signal sequence, the recombinant protein is secreted into the periplasmic space. The signal sequence is removed after the membrane passage.
- pQTK_1 the bacteria are extracted with PBS / 8 M urea. After sonication, the suspension is centrifuged.
- the fusion protein is purified from the supernatant by metal chelate affinity chromatography using a Ni-NTA agarose matrix (Qiagen), as described by Hochuli et al. (J Chromatogr 411: 177-184; 1987).
- the extract is placed on the column; it is then washed with PBS / urea and the fusion protein is eluted from the column with 150 mM imidazole in PBS / urea.
- the collagen preparations are characterized by SDS-PAGE, determination of the amino acid composition, the isoelectric point and by electron microscopy.
- the molecular weights can be determined by SDS-PAGE.
- the molecular weight of the protein obtained after expression of the cDNA amplified using the above primers is ⁇ 28.5 kDa.
- the isoelectric point can be determined by titration in aqueous solution.
- the IEP of sponge collagen is usually at pH 6.5 - 8.5 (for comparison, IEP of bovine collagen: pH 7.0 ⁇ 0.09). That from SEQ ID No. 8 cDNA-derived peptide shown (see SEQ ID No. 7) has a predicted isoelectric point of 8.185.
- the charge at pH 7.0 is 4,946.
- Amino acid composition The determination of the amino acid composition can be carried out with the help of an automatic amino acid analyzer.
- Electron microscopy The electron-microscopic characterization of the isolated sponge collagen can be carried out by transmission electron microscopy (TEM). For this purpose, the freeze-dried collagen sample is contrasted negatively with a 2% phosphotungstic acid (Harris, Negative staining and cryoelectron microscopy. Royal Microscopical Society Microscopy Handbook No 35.BIOS Scientific Publishers Ltd, Oxford, UK).
- TEM transmission electron microscopy
- silicatein activity (silicatein- ⁇ and silicatein- ⁇ ) has been described (PCT / US99 / 30601; DE 10037270 A 1; PCT / EP01 / 08423; DE 103 52 433.9).
- the silica can e.g. B. with the help of a molybdate-based detection method, such as. B. the colorimetric "Silicon Test" (Merck; 1.14794) can be determined quantitatively.
- the amount of silica can be calculated from the absorbance values at 810 nm using a calibration curve with a silicon standard (Merck 1.09947).
- silica is in the form of a metasilicate (sodium salt or salt of another alkali, alkaline earth or metal ion), silicon Complex (which is in equilibrium with free orthosilicic acid or orthosilicate; for example silicon catecholate [dipotassium tricatecholatosilicon] or in the form of orthosilicic acid or an orthosilicate in a suitable buffer (for example 50 mM Tris-HCl pH 7.0, 100 mM NaCl, 0 , 1 mM ZnS0 4 and 0.1 mM ß-mercaptoethanol or other buffer; the presence of Zn is advantageous in the incubation with silicase or carbonic anhydrases, which are Zn enzymes) over a for the desired amount of the silica product formed (amorphous Silicon dioxide) adapted period with a template and an enzyme.
- the incubation can be carried out at different temperatures. Room temperature (22 ° C.) has proven to be advantageous, but also higher (eg
- the metasilicate can either be dissolved in the buffer used or previously (possibly as a higher concentrated stock solution) in an alkaline solution (such as 0.01 N NaOH). In the latter case, the metasilicate solution obtained must be neutralized (advantageous pH: 7.2).
- the template is one or more different molecules, molecular aggregates or surfaces which have functional groups which interact with orthosilicic acid, oligomeric or polymeric silicic acids and their salts (orthosilicates, metasilicates).
- the molecules containing the hydroxyl groups are collagen or a silicatein (see FIGS. 7-10).
- the collagen is a collagen from a sponge, in particular a collagen according to SEQ ID No. 7 or a polypeptide homologous thereto, which has at least 25%, preferably at least 50%, in its amino acid sequence. more preferably has at least 75% and most preferably at least 95% sequence identity to the sequence shown in SEQ ID No. 7, or parts thereof.
- the collagen specified in SEQ ID No. 7 is a non-fibrillary collagen (collagen 3) the sea sponge S. domuncula. This collagen was found to be more efficient than fibrillar bovine collagen (see Figure 10).
- the silicatein is a silicatein from a sponge according to SEQ ID No. 3 or a polypeptide homologous to it which has at least 25%, preferably at least 50%, more preferably at least 75 in its amino acid sequence % and most preferably at least 95% has sequence identity to the sequence shown in SEQ ID No. 3, or parts thereof (see FIGS. 7-10).
- silicatein- ⁇ SEQ ID No. 3
- silicatein- ⁇ SEQ ID No. 5
- a polypeptide homologous to it can also be active, the amino acid sequence of which is at least 25%, preferably at least 50%, more preferably at least 75% and am most preferably has at least 95% sequence identity to the sequence shown in SEQ ID No. 5, or parts thereof are used.
- a mixture of one or more templates can also be used (see FIGS. 7-10).
- the collagen from a sponge according to SEQ ID No. 7 or a polypeptide homologous thereto, which in its amino acid sequence has at least 25%, preferably at least 50%, more preferably at least 75% and most preferably at least 95% sequence identity to that in SEQ ID No. 7, or parts thereof, can be provided both in vivo, in a cell extract or lysate or in purified form.
- the enzyme is a polypeptide of a silicase from Sube tes domuncula according to SEQ ID No. 1 or a homologous polypeptide which in the amino acid sequence of the carbonic anhydrase domain is at least 25%, preferably at least 50%, more preferably at least 75% and most preferably has at least 95% sequence identity to the sequence shown in SEQ ID No. 1, a metal complex of the polypeptide, or parts thereof (see Figures 7-10).
- polypeptide homologous thereto which is in the amino acid sequence of the carbonic anhydrase domain at least 25%, preferably at least 50%, more preferably at least 75% and most preferably at least 95% sequence identity to the sequence shown in SEQ ID No. 1 can be provided both in vivo, in a cell extract or lysate or in purified form.
- the amount of insoluble Si0 2 formed increases with increasing concentration of carbonic anhydrase (see FIGS. 8 and 9).
- the amount of Si0 2 formed depends on the concentration of the template used; an increase is found with increasing concentration of, for example, silicatein- ⁇ (see FIG. 8) or collagen (see FIG. 9).
- the incubation with silicatein and carbonic anhydrase can be carried out simultaneously (see FIG. 9) or in succession (see FIGS. 7, 8 and 10).
- biomaterials and composite materials can serve as templates for the formation of silica, such as fibrillary chitin, which is obtained by a process described (DE 102 10 571.5.
- Composition and process for producing modified fibrillar chitin and potentiating additives) biologically highly active preparations and their use as protection and food supplements during prenatal and postnatal development and adult life phases in humans and animals.
- Applicant and inventor Müller WEG, Schröder HC, Lorenz B, Senyuk OF, Gorowoj LF).
- the method is also suitable for the synthesis of other polymeric metal (IV) compounds from purely inorganic metal (IV) compounds, where also (1) a template (molecule, molecular aggregate or surface) and (2) a polypeptide or a metal complex of a polypeptide is used for the synthesis, which is either characterized in that the polypeptide comprises an animal, plant, bacterial or fungal carbonic anhydrase domain which has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1, or in that the polypeptide an animal, bacterial, vegetable or fungal silicatein- ⁇ -domain or silicatein- ⁇ -domain which comprises at least 25%, preferably at least 50%, more preferably at least 75% and most preferably at least 95% sequence identity to the sequence shown in SEQ ID No. 3 or sequence shown in SEQ ID No. 5.
- a template molecule, molecular aggregate or surface
- a polypeptide or a metal complex of a polypeptide is used for the synthesis, which is either characterized in that the polypeptide comprises an
- the material (or the reaction mixture) can be centrifuged off in a table centrifuge (12,000 x g; 15 min; 4 ° C), washed with ethanol and air-dried. The sediment can then be hydrolyzed with 1 M NaOH. In the resulting solution, the released silicate is using a molybdate-based detection method, such as. B. the colorimetric "Silicon Test" from Merck, measured quantitatively.
- the detection of the silica product formed can also be carried out using a "High Performance Field Emission Electron Probe Microanalyzer (EPMA)".
- EPMA Electron Probe Microanalyzer
- a JXA-8900RL Electron Probe Microanalyzer (JEOL, Inc., Peabody, MA, USA) was used for the experiment shown in FIG. This device combines high resolution scanning electron microscopy (SEM) with high quality X-ray analysis.
- biomaterials which either consist of the template materials mentioned (hydroxyl group-containing molecules) themselves or are coated with them. These can also be surfaces of glass, metals, metal oxides, plastics, biopolymers or other materials.
- a literature overview of surface-modified biomaterials can be found in: Ratner BD et al (ed.) Biomaterials Science - An Introduction to Materials in Medicine. Academic Press, San Diego, 1996.
- the conditions used in conventional physical / chemical methods to make these modifications often have a deleterious (destructive) effect on the biomaterials.
- the method according to the invention uses "mild" conditions which protect the biomaterials, since it is based solely on biochemical / enzymatic reactions.
- the method according to the invention is also used in the production of surface modifications (coating) of collagen, which serves as tissue, bone or denture material, and of collagen fleeces ("tissue engineering").
- the surface modifications serve to increase the stability and porosity and to improve the resorbability.
- sponge collagen As with other collagens, the advantages of sponge collagen as a biomaterial are biodegradability and low toxicity and immunogenicity.
- sponge collagen does not have the disadvantages of collagen previously obtained primarily from animal skins and bones of pigs, calves and cattle, in which the possibility of infection by pathogenic germs cannot be excluded.
- Another advantage of the process is that no organic solvents have to be used to dissolve the starting substrates used (silicas and metasilicates and their salts), as is the case with organic silicon compounds (e.g. TEOS). This prevents damage to the biopolymers to be modified, such as collagen.
- silica amorphous silicon dioxide
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP05746455A EP1740707A1 (de) | 2004-04-30 | 2005-05-02 | Enzym- und template-gesteuerte synthese von silica aus nicht-organischen siliciumverbindungen sowie aminosilanen und silazanen und verwendung |
JP2007509991A JP2007535319A (ja) | 2004-04-30 | 2005-05-02 | 酵素とテンプレートで制御された、非有機シリコン化合物、アミノシラン、シラザンからのシリカの合成、並びにその使用 |
US11/579,019 US20080293096A1 (en) | 2004-04-30 | 2005-05-02 | Enzyme and Template-Controlled Synthesis of Silica from Non-Organic Silicon Compounds as Well as Aminosilanes and Silazanes and Use Thereof |
CA002565118A CA2565118A1 (en) | 2004-04-30 | 2005-05-02 | Enzyme and template-controlled synthesis of silica from non-organic silicon compounds as well as aminosilanes and silazanes and use thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102004021230A DE102004021230A1 (de) | 2004-04-30 | 2004-04-30 | Enzym-und Template-gesteuerte Synthese von Silica aus nicht-organischen Siliciumverbindungen sowie Aminosilanen und Silazanen und Verwendung |
DE102004021230.9 | 2004-04-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005106003A1 true WO2005106003A1 (de) | 2005-11-10 |
Family
ID=34969119
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP2005/004734 WO2005106003A1 (de) | 2004-04-30 | 2005-05-02 | Enzym- und template-gesteuerte synthese von silica aus nicht-organischen siliciumverbindungen sowie aminosilanen und silazanen und verwendung |
Country Status (6)
Country | Link |
---|---|
US (1) | US20080293096A1 (de) |
EP (1) | EP1740707A1 (de) |
JP (1) | JP2007535319A (de) |
CA (1) | CA2565118A1 (de) |
DE (1) | DE102004021230A1 (de) |
WO (1) | WO2005106003A1 (de) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1983054A1 (de) * | 2007-04-20 | 2008-10-22 | Georg-August-Universität Göttingen | Kohlenstoffanhydrase und Verfahren zu ihrer Verwendung |
WO2013098164A3 (de) * | 2011-12-30 | 2013-10-03 | Leibniz-Institut Für Neue Materialien Gemeinnützige Gmbh | Neue organisch-anorganische kompositmaterialien durch biomineralisation |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2379729A2 (de) | 2008-12-19 | 2011-10-26 | Novozymes A/S | Verwendung von enzymen mit silicase-aktivität |
EP2489346A1 (de) | 2011-01-26 | 2012-08-22 | NanotecMARIN GmbH | Nahrungsergänzungsmittel und injizierbares Material zur Prophylaxe und Behandlung von Osteoporose und anderen Knochenerkrankungen |
US8319181B2 (en) | 2011-01-30 | 2012-11-27 | Fei Company | System and method for localization of large numbers of fluorescent markers in biological samples |
JP2016158544A (ja) * | 2015-02-27 | 2016-09-05 | 国立大学法人鳥取大学 | 新規タンパク質 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000035993A1 (en) * | 1998-12-18 | 2000-06-22 | The Regents Of The University Of California | Methods, compositions, and biomimetic catalysts for in vitro synthesis of silica, polysilsequioxane, polysiloxane, and polymetallo-oxanes |
WO2002010420A2 (de) * | 2000-07-28 | 2002-02-07 | Mueller Werner E G | Silicatein-vermittelte synthese von amorphen silikaten und siloxanen und ihre verwendung |
WO2004033679A1 (de) * | 2002-10-03 | 2004-04-22 | Johannes Gutenberg-Universität Mainz | Abbau und modifizierung von silicaten und siliconen durch silicase und verwendung des reversiblen enzyms |
-
2004
- 2004-04-30 DE DE102004021230A patent/DE102004021230A1/de not_active Withdrawn
-
2005
- 2005-05-02 JP JP2007509991A patent/JP2007535319A/ja active Pending
- 2005-05-02 US US11/579,019 patent/US20080293096A1/en not_active Abandoned
- 2005-05-02 CA CA002565118A patent/CA2565118A1/en not_active Abandoned
- 2005-05-02 EP EP05746455A patent/EP1740707A1/de not_active Withdrawn
- 2005-05-02 WO PCT/EP2005/004734 patent/WO2005106003A1/de not_active Application Discontinuation
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000035993A1 (en) * | 1998-12-18 | 2000-06-22 | The Regents Of The University Of California | Methods, compositions, and biomimetic catalysts for in vitro synthesis of silica, polysilsequioxane, polysiloxane, and polymetallo-oxanes |
WO2002010420A2 (de) * | 2000-07-28 | 2002-02-07 | Mueller Werner E G | Silicatein-vermittelte synthese von amorphen silikaten und siloxanen und ihre verwendung |
WO2004033679A1 (de) * | 2002-10-03 | 2004-04-22 | Johannes Gutenberg-Universität Mainz | Abbau und modifizierung von silicaten und siliconen durch silicase und verwendung des reversiblen enzyms |
Non-Patent Citations (1)
Title |
---|
WEAVER JAMES C ET AL: "Molecular biology of demosponge axial filaments and their roles in biosilicification.", MICROSCOPY RESEARCH AND TECHNIQUE. 1 NOV 2003, vol. 62, no. 4, 1 November 2003 (2003-11-01), pages 356 - 367, XP008051925, ISSN: 1059-910X * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1983054A1 (de) * | 2007-04-20 | 2008-10-22 | Georg-August-Universität Göttingen | Kohlenstoffanhydrase und Verfahren zu ihrer Verwendung |
WO2013098164A3 (de) * | 2011-12-30 | 2013-10-03 | Leibniz-Institut Für Neue Materialien Gemeinnützige Gmbh | Neue organisch-anorganische kompositmaterialien durch biomineralisation |
Also Published As
Publication number | Publication date |
---|---|
DE102004021230A1 (de) | 2005-11-24 |
US20080293096A1 (en) | 2008-11-27 |
CA2565118A1 (en) | 2005-11-10 |
EP1740707A1 (de) | 2007-01-10 |
JP2007535319A (ja) | 2007-12-06 |
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