EP1740707A1

EP1740707A1 - Enzyme and template-controlled synthesis of silica from non-organic silicon compounds as well as aminosilanes and silazanes and use thereof

Info

Publication number: EP1740707A1
Application number: EP05746455A
Authority: EP
Inventors: Heiko Schwertner; Werner E.G. Müller; Heinz C. SCHRÖDER
Original assignee: Schwertner Heiko Dr
Current assignee: Schwertner Heiko Dr
Priority date: 2004-04-30
Filing date: 2005-05-02
Publication date: 2007-01-10
Also published as: WO2005106003A1; JP2007535319A; US20080293096A1; CA2565118A1; DE102004021230A1

Abstract

The present invention relates to a method for synthesis of amorphous silicon dioxide (silica, condensation products of silicic acid) and other polymeric metal (IV) compounds from non-organic silicon compounds or metal (IV) compounds as well as from aminosilanes and silazanes, whereby (1) a template (collagen or another molecule, interacting with orthosilicic acid or polymeric silicic acid and salts thereof or other metal (IV) compounds) and (2) a silicase/carbonic anhydrase or a silicatein or similar polypeptide are used for synthesis. Said invention also relates to the technical use thereof.

Description

Enzvm- and template-controlled synthesis of silica from non-organic silicon compounds as well as aminosilanes and silazanes and use

description

1. State of the art

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).

Orthosilicic acid (H Si0 ₄ ) is a very weak acid. Diluted solutions are only stable for some time at low pH values (pH 2-3). When the pH is increased or decreased, intermolecular elimination of water (condensation) occurs, the first condensation product being silicic acid (pyro silicic acid; HβSi ₂ 0). Cyclic silicas as well as cage-like silicas and polysilicic acids are initially formed as further condensation products - with a smaller number of links (n = 3, 4 or 6). These metasilicas have the gross composition (H ₂ Si0 ₃ ) _n . 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. For all silicas, the silicon atoms are at the center of regular tetrahedra, the corners of which form four oxygen atoms. In the case of polysilicic acids or in amorphous silicon dioxide, the oxygen atoms simultaneously belong to the neighboring, irregularly linked tetrahedra. 1.1. biosilica

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). In addition, 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.

Two enzymes that are involved in the synthesis of the Si0 ₂ skeleton in silicate-forming organisms and their technical use have been described. The first enzyme is silicatein, which occurs in three forms, silicatein-α, -ß and - γ (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. Inventors / Applicants: Morse DE , Stucky GD, Deming, TD, Cha J, Shimizu K, Zhou Y; DE 10037270 A1. Silicatein-mediated synthesis of amorphous silicates and siloxanes and their use. German Patent Office 2000. Applicant and inventor: Müller WEG, Lorenz B, Krasko A , Schröder HC; PCT / EP01 / 08423. Silicatein-mediated synthesis of amorphous silicates and siloxanes and use thereof. Inventors / Applicants: Müller WEG, Lorenz B, Krasko A, Schröder HC). Silicatein-α 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-α 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).

According to the state of the art, both silicateine, silicatein-α and silicatein-β 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. Inventors: Müller WEG, Krasko A, Schröder HC; Schröder HC, Krasko A, Le Pennec G, Adell T, Wiens M, Hassanein H, Müller IM, Müller WEG (2003) 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. In addition, the silicase has the ability to dissolve lime material in analogy to the carbonic anhydrase.

So far it has not been disclosed that 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. 1.2. collagen

In addition to elastin, polyanionic proteoglycans and structural glycoproteins, 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.

In vertebrates, 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 (α 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 α-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 (α1, α2, α3). 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 α 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.

In the so-called minority collagens, the triple helix is only found in some sections of the molecule; other sections have globular domains. They include collagen types IV to XIX. The type V and type XI minority collagens, however, also form fibril structures. 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.

1.2.1. Cloning and sequencing of collagen from sponges

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). Electron microscopic examinations of the collagen from the sea sponge Geodia cydonium show 20 to 25 nm thick collagen fibrils with a periodicity of 19.5 nm (Diehl-Seifert et al. (1985) J Cell Sei 79: 271-285; Gramzow et al. (1988) J Histochem Cytochem 36: 205-212). The collagen that we cloned from the sea sponge S. domuncula (Schröder et al. (2000) FASEB J 14: 2022-2031) consists of (i) a non-collagenic Λ / -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. In contrast, 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.

The expression of collagen in sponge cells (primmorphs, a special form of 3D cell aggregates formed from single sponge cells; DE 19824384 were used. Production of primmorphs from dissociated cells of sponges, corals and other invertebrates: methods for culturing cells of sponges and further invertebrates for the production and detection of bioactive substances, for the detection of environmental toxins and for the cultivation of these animals in aquariums and in the field Inventor and applicant: Müller WEG, Brummer F; Müller et al. (1999) Mar Ecol Prog Ser 178: 205- 219) is stimulated by myotrophin (Schröder et al. (2000) FASEB J 14: 2022-2031; Krasko et al. (2000) Eur J Biochem 267: 4878-4887). Myotrophin is a growth-promoting protein that was also cloned from S. domuncula by the inventors.

2. Subject of the invention

Surprisingly, it has now been possible for the inventors to show that the 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. So far, it was only known that 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. Ed.] 38: 780-782; PCT / US99 / 30601; DE 10037270 A 1; PCT / EP01 / 08423). Enzymes containing the carbonic anhydrase domains were known to be able to remove inorganic polysilicates (polysilicic acid). ren) and amorphous and also crystalline silicon dioxide with the release of silica (Schröder et al. (2003) Prog Mol Subcell Biol 33: 250-268; DE 102 46 186.4; PCT / EP03 / 10983), but not for the catalysis of a template-controlled Synthesis of amorphous silicon dioxide (silica) from orthosilicates and metasilicates.

According to a first aspect of the present invention, 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. 1 sequence, or in that the polypeptide comprises an animal, bacterial, plant or fungus silicatein-α-domain or silicatein-β-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.

Also preferred is 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.

Also preferred is 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.

According to a further aspect of the present invention, 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. 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

[www.ncbi.nlm.nig.gov]) is found in the sponge silicase in the amino acid range from aa ₈₇ to aa ₃₃₅ . Most of the characteristic amino acids that make up the eukaryotic type carbonic anhydrase signature (Fujikawa-Adachi et al. (1999) Biochim Biophys Acta 1431: 518-524; Okamoto et al. (2001) Biochim Biophys Acta 1518: 311-316 ) are also present in the sponge silicase.

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).

In addition to the sponge silicase, carbonic anhydrases from other organisms (some of which are commercially available) can also be used in the process according to the invention.

The invention will now be described in more detail below by means of the attached examples, but without being restricted thereto. The attached sequences and figures show:

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-α from S. domuncula (SIA_SUBDO) used according to the invention.

SEQ ID No. 4: The nucleic acid sequence of the silicate one-α from S. domuncula used according to the invention.

SEQ ID No. 5: The amino acid sequence of the silicate one-β from S. domuncula (SIA_SUBDO) used according to the invention.

SEQ ID No. 6: The nucleic acid sequence of the silicate one-β 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.

Figure 1:

A. Electron micrographs of isolated collagen from Geodia cydonium. (Aa) Bundle of collagen fibrils. (Ab) Negatively colored fibrils. B. scanning electron micrographs of sponge Si0 ₂ skeleton elements. Top left to right: Tylostyle (Suberites domuncula), Spheraster (Geodia cydonium), Sterraster (Geodia cydonium). Bottom left to right: Sterraster (Geodia cydonium) in increasing magnification.

Figure 2:

Nucleotide sequence of the carbonic anhydrase (silicase) clone (S. domuncula) as well as forward primer (positive-1 and positive-2) and reverse primer (negative-1) for the amplification of the coding for the long and the short silicase form cDNA for cloning into the expression vector pGEX-4T-2 and amino acid sequence of the recombinant proteins (long and short form of the silicase). Protein information about the proteins is: Protein information about CAexpresL.prt (long form): Molecular weight: 43130.74 Dalton + 25000 Da GST ~~> 68 kDa

379 amino acids46 strongly basic (+) amino acids (K, R) 46 strongly acidic (-) amino acids (D, E) 120 hydrophobic amino acids (A, I, L, F, W, V) 103 polar amino acids (N, C, Q , S, T, Y) 7.666 Isoelectric point 2.871 charge at pH 7.0

Protein information about CAexpresS.PRO (1, 2- "4) (short form): Molecular weight: 32271, 28 Dalton + 25000 Dalton GST ~~> 57 kDa284 amino acids 35 Strongly basic (+) amino acids (K, R) 39 Stark acidic (-) amino acids (D, E) 91 hydrophobic amino acids (A, I, L, F, W, V) 70 polar amino acids (N, C, Q, S, T, Y) 6,701 isoelectric point -1, 795 charge at pH 7.0

Figure 3:

Nucleotide sequence of the carbonic anhydrase (silicase) clone (S. domuncula) as well as forward primer (positive-1 and positive-2) and reverse primer (negative-1) for the amplification of the coding for the long and the short silicase form cDNA for cloning into the expression vector pBAD / glll A and amino acid sequence of the recombinant

Proteins (long and short form of silicase). Protein information about the proteins is:

Long form: molecular weight: 48430.78 daltons

424 amino acids

49 strongly basic (+) amino acids (K, R)

53 strongly acidic (-) amino acids (D, E)

137 hydrophobic amino acids (A, I, L, F, W, V)

111 polar amino acids (N, C, Q, S, T, Y)

7.005 isoelectric point

0.045 charge at pH 7.0

Short form: molecular weight: 33,702.52 daltons

330 amino acids

0.045 charge at pH 6.52

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 Λ / 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 Λ / col and / - / ^' ndlll are marked); amino acid sequence of the recombinant protein derived from the nucleotide sequence.

Figure 5:

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). Conserved amino acid residues (similar or related in terms of their physicochemical properties) in the sequences are shown in white on black. NC1: non-collagenic Λ / -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.

Figure 6:

A. Preparation of recombinant silicatein-α. B. Production of Recombinant Silicase.

Figure 7:

In the experiment shown here, 100 μM Na-metasilicate in the absence or presence of 20 μg / ml recombinant silicatein-α or bovine serum albumin (BSA) in buffer (50 mM Tris-HCl pH 7.0, 100 mM NaCl, 0.1 mM ZnS0 ₄ and 0.1 mM β-mercaptoethanol) incubated for 10 min at room temperature. Then - as indicated in the figure - 4 μg / ml recombinant sponge collagen, 10 μg / ml carbonic anhydrase (from bovine erythrocytes) and / or 10 mM catechol were added and incubated for a further 2 h at room temperature. All stated concentrations are the final concentrations after adding all components to the batches. To detect the amorphous silicon dioxide formed, the Centrifuged reaction batches in a table centrifuge (12,000 xg; 15 min; 4 ° C), washed with ethanol and air dried. The sediments were then hydrolyzed with 1 M NaOH and the silicate released was measured quantitatively using a molybdate-based detection method (colorimetric "silicone test" from Merck).

The experiment shows that maximum amounts of amorphous silica are synthesized in the presence of collagen, silicatein-α and carbonic anhydrase (0.098 - 0.117 OD units) and in the presence of collagen and silicatein-α (0.138 OD units). Smaller amounts of insoluble Si0 ₂ were determined in the absence of carbonic anhydrase (0.057 OD units) and in the absence of silicatein-α (0.037 and 0.048 OD units). In the absence of collagen, only very small amounts of insoluble SiO ₂ (0.014-0.019 or 0.022 or 0-0.018 or 0.008 OD units) were measured either with or without silicatein or carbonic anhydrase or both enzymes. Also in the presence of collagen alone, only a little insoluble Si0 _{2 was} formed (0.008 and 0.032 OD units). In the presence of BSA instead of silicatein and collagen, only very small amounts of Si0 _{2 were} measured (0.015 OD units) both with and without carbonic anhydrase. The addition of catechol led to a reduction in the amount of insoluble SiO ₂ .

Figure 8:

In the experiment shown here, 100 μM Na metasilicate in the absence or presence of 20 to 400 μg / ml recombinant silicatein-α or bovine serum albumin (BSA; 20 μg / ml) in buffer (50 mM Tris-HCl pH 7.0, 100 mM NaCl, 0.1 mM ZnS0 ₄ and 0.1 mM β-mercaptoethanol) for 10 min at room temperature. Then - as indicated in the figure - 4 μg / ml recombinant sponge collagen, 10 μg / ml carbonic anhydrase (bovine erythrocytes) and / or 10 mM catechol were added and incubated for a further 5 h at room temperature. All stated concentrations are the final concentrations after adding all components to the batches. To detect the amorphous silicon dioxide formed, the reaction batches were treated further as described in FIG. 5 and the amount of insoluble SiO _{2 formed was} determined. It was found that the amount of insoluble Si0 _{2 increased} with increasing concentration of carbonic anhydrase (from 0.002 to 0.050 OD units). A pre-incubation with silicatein-α (10 min) did not lead to any further increase, but to a decrease in Si0 ₂ formation (0.015 and 0.030) under the conditions used. In the presence of BSA instead of silicatein and collagen, only very small amounts of Si0 _{2 were} measured (0.020 OD units OD units). Without the addition of catechol, the amounts of insoluble SiO _{2 formed were} greater.

Figure 9:

In the experiment shown here, 100 μM Na metasilicate and 4 μg / ml recombinant sponge collagen were used in the presence of increasing concentrations (2 to 20 μg / ml) of carbonic anhydrase (from bovine erythrocytes) in buffer (50 mM Tris HCl pH 7.0, 100 mM NaCl, 0.1 mM ZnS0 ₄ and 0.1 mM β-mercaptoethanol) in the presence of 10 mM catechol for 2 h at room temperature. Here, the amount of insoluble Si0 ₂ formed increased sharply (from 0.015 to 0.060 OD units). Likewise, the amount of SiO ₂ formed increased sharply with an increasing amount of collagen (1.2 to 10 μg / 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). In the presence of bovine serum albumin (BSA; 20 μg / ml) instead of collagen, 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.

Figure 10:

In the experiment shown here, 100 μM Si-catecholate complex in the absence or presence of 20 μg / ml recombinant silicatein-α in buffer (50 mM Tris-HCl pH 7.0, 100 mM NaCl, 0.1 mM ZnS0 ₄ and 0.1 mM β-mercaptoethanol) for 10 min at room temperature. Then - as indicated in the figure - either recombinant sponge collagen (1 to 4 μg / ml) or purified bovine collagen (2 to 10 μg / ml) and 10 μg / ml carbonic anhydrase (from bovine erythrocytes) added and incubated for a further 3 h at room temperature. 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 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. As with the use of Na metasilicate (see FIG. 7), the amount of SiO ₂ formed increased sharply with increasing amount of sponge collagen (1 to 4 μg / ml) (from 0.002 to 0.010 OD units). Similar to the results obtained with Na metasilicate (see FIG. 5), the SiO ₂ formation was lower in the presence of catechol, which can be explained by a shift in the equilibrium towards the Si catecholate complex. In the presence of the carbonic anhydrase alone, no insoluble Si0 _{2 was} formed (not shown in the figure). Increasing the concentration of recombinant silicatein-α to 40 and 400 μg / ml led to a decrease in Si0 ₂ formation (not shown in the figure).

Figure 11:

The evidence of the silica products formed is shown with the aid of a "High Performance Field Emission Electron Probe Microanaiyzer (EPMA)". The incubation was carried out in the absence (= control) or in the presence of 50 μg / ml carbonic anhydrase (from bovine erythrocytes; Calbiochem company) and 30 μg / ml collagen in buffer (50 mM Tris-HCl pH 7.0, 100 mM NaCl, 0.1 mM ZnS0 ₄ and 0.1 mM β-mercaptoethanol) with 1 mM Na metasilicate at room temperature. The incubation period was 4 hours. 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).

3. Production and verification of the components required for the process

3.1. Production of the silicase The purification of the silicase from natural sources such as tissues or cells and the recombinant production of the enzyme have been described and are state of the art (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.Inventors: Müller WEG, Krasko A, Schröder HC).

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 ntι ₂₂ - ntι ₂₄ to ntι ₂₅₉ - ntι ₂₆ i (stop codon); Length of the polypeptide: 379 amino acids; molecular weight (M _r ) of the polypeptide: 43131; isoelectric point (pl): 6.5.

For the experiments described here, the recombinant S. domuncula silicase was produced as a glutathione S-transferase (GST) fusion protein. 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.

Another alternative is the production of the recombinant silicase in E. coli using the oligo-histidine expression vector pBAD / gl | IA (Invitrogen), in which the recombinant protein is secreted into the periplasmic space on the basis of the gene III signal sequence (Figure 3). 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.

It is also possible to use an insert which comprises the entire derived silicone protein (long form).

3.2. Manufacture of silicatein

The purification of the silicatein-α and the silicatein-β from natural sources such as tissues or cells and the recombinant production of the enzymes have been described and are state of the art (DE 10037270 A 1. Silicatein-mediated synthesis of amorphous silicates and siloxanes and their use. German Patent Office 2000. Applicant and inventor: Müller WEG, Lorenz B, Krasko A, Schröder HC; PCT / EP01 / 08423. Silicatein-mediated synthesis of amorphous silicates and siloxanes and use thereof. Inventors / Applicants: Müller WEG, Lorenz B , Krasko A, Schröder HC; 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).

For the experiments described here, the recombinant silicatein-α 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-α 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: Λ / col and Xba \). After transformation of E. coli XL1-Blue, the expression of the fusion protein was induced with L-arabinose. 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.

It is also possible to use an insert which comprises the entire derived silicacein α-protein (long form).

3.3. Production of sponge collagen

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 ,

3.3.1. Isolation of native sponge collagen

A simple process for isolating collagen from various marine sponges has been described (DE 100 10 113 A 1. Process for isolating sponge collagen and producing nanoparticulate collagen. Applicant: W. Schatton. Inventor: Kreuter J, Müller WEG, Schatton W, Swatschek D, Schatton M; Swatschek et al. (2002) Eur J Pharm Biopharm 53: 107-113). The sponge collagen is obtained with a high yield (> 30%).

3.3.2. Production of recombinant sponge collagen

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). Can serve as primers for the PCR (with subsequent use of pBAD / glllA); Forward primer: TAT cc atg gTG GCA ATA TCA GGT CAG GCT ATA GGA CCT C and reverse primer: TAT AA GC TT CGC TTT GTG CAG ACA ACA CAG TTC AGT TC; Restriction nucleases for insertion into the expression vector: Λ / col and - // ndlll. After transformation of Escherichia co // strain XL1-Blue with the plasmid (expression vector), expression of the fusion protein is induced with L-arabinose (for pBAD / glllA) or with isopropyl-β-D-thiogalactopyranoside (IPTG; for pQTK_1) , 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. When using 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.

Molecular weight, isoelectric point: 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 (IEP) 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).

3.4. Detection of silicase activity

The method for detecting the silicase activity of (commercial) carbonic anhydride preparations (eg from bovine erythrocytes; Calbiochem company) or the recombinant sponge silicase has been described (DE 102 46 186.4; PCT / EP03 / 10983 ).

3.5. Detection of silicatein activity

The method for the detection of silicatein activity (silicatein-α and silicatein-β) 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).

4. Description of the method of silica synthesis

In the process according to the invention, 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 37 ° C.) or lower temperatures (eg B. 15 ° C) were used successfully.

For this purpose, 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).

It has proven to be advantageous if the molecules containing the hydroxyl groups are collagen or a silicatein (see FIGS. 7-10).

It has turned out to be particularly advantageous if 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).

It has also proven to be particularly advantageous if 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).

In addition to silicatein-α (SEQ ID No. 3), silicatein-β (SEQ ID No. 5) or 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 (for example collagen and silicatein) 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). The polypeptide of a S. domuncula silicase according to SEQ ID No. 1 or a 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.

It is also advantageous to use commercial carbonic anhydrases, such as the carbonic anhydrase from bovine erythrocytes (see FIGS. 7-10).

The addition of catechol, which complexes free silica, leads to a reduction in the amount of insoluble SiO ₂ (see FIGS. 7 and 10).

Maximum amounts of amorphous silica are synthesized in the presence of collagen, silicatein and carbonic anhydrase as well as in the presence of collagen and silicatein (see Figure 7). Smaller amounts of insoluble Si0 ₂ are obtained in the presence of collagen and carbonic anhydrase (see Figure 7). Depending on the conditions used (incubation time), preincubation with silicatein can also lead to a decrease in Si0 ₂ formation (see FIG. 8). In the absence of collagen, only very small amounts of insoluble SiO _{2 are} formed with silicatein or carbonic anhydrase or both enzymes (see FIG. 7). Likewise, only slightly insoluble SiO _{2 is} formed in the presence of collagen (see FIG. 7). Control experiments with BSA instead of silicatein and collagen as a template show only a very slight formation of insoluble SiO ₂ (see FIGS. 7-9).

The amount of insoluble Si0 _{2 formed} increases with increasing concentration of carbonic anhydrase (see FIGS. 8 and 9).

Furthermore, 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-α (see FIG. 8) or collagen (see FIG. 9).

An increase in the Na metasilicate concentration did not lead to any further increase, but rather to a decrease in the SiO ₂ formation (see FIG. 9). In addition to metasilicates, silicon complexes (for example the silicon-catechol complex) can also be used; here too the amount of Si0 ₂ formed increases with the amount of collagen (see FIG. 10). However, when using the silicon-catechol complex instead of metasilicate, the yields of insoluble SiO _{2 formed} are lower (cf. FIGS. 7-9 and FIG. 10).

The use of other silicon complexes such as the silicon complexes with gallic acid or tropolon (tristropolonato silicon chloride) is also possible.

The incubation with silicatein and carbonic anhydrase can be carried out simultaneously (see FIG. 9) or in succession (see FIGS. 7, 8 and 10).

In addition to collagen, a number of other 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-α-domain or silicatein-β-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. 4.1. Detection of the silicon dioxide formed

To detect the products (amorphous silicon dioxide formed), 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 (element analysis) can also be carried out using a "High Performance Field Emission Electron Probe Microanalyzer (EPMA)". 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.

The approaches for analysis using the "High Performance Field Emission Electron Probe Microanalyzer" contained 1 mM Na metasilicate in 50 mM Tris-HCl pH 7.0, 100 mM NaCI, 0.1 mM ZnS0 ₄ and 0.1 mM ß- mercaptoethanol. The incubation was carried out in the absence (= controls) or in the presence of 50 μg / ml carbonic anhydrase (from bovine erythrocytes; Calbiochem company) and 30 μg / ml collagen for 4 h at room temperature.

100 μl of the samples (batches after incubation) were applied to the supports. The carrier with the preparations was subjected to Kohlenstoffbedampfung (Emitech K959) under vacuum (10 mbar ^"4). In addition to Si Ca, Na and Cl were determined.

The results showed that a clear formation of silicon aggregates was detectable in the batches with carbonic anhydrase and collagen, but not in the controls (absence of carbonic anhydrase and collagen) (see FIG. 11). There were no matches in the localization of the signals for Si, Ca, Na and Cl.

5. Uses of the procedure

The described process for the enzymatic synthesis of amorphous silica from inorganic (non-organic) silicon compounds has a number of different industrial and technical uses, namely:

1.) Use for the surface modification of 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. In comparison to the conventional methods, the method according to the invention uses "mild" conditions which protect the biomaterials, since it is based solely on biochemical / enzymatic reactions. In particular, 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.

As with other collagens, the advantages of sponge collagen as a biomaterial are biodegradability and low toxicity and immunogenicity. However, 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.

2.) Use for modification or for the synthesis of nano-structures from silica (amorphous silicon dioxide). Using the method according to the invention, it is possible to synthesize defined two- and three-dimensional structures from silica (or other polymeric metal (IV) compounds) on a nano scale from purely inorganic starting substrates (silicic acid, metasilicic acid and their salts). The structures formed can be used in nanotechnology.

3.) Use of the method according to the invention for the production of three-dimensional, silica-coated matrices from collagens with defined physicochemical properties for the production of tissues / organs of the human organism with the body's own cells, which are used as replacement tissue for the treatment of oncological defects, post-traumatic organ and tissue damage, burn injuries , Vascular occlusions and surgical wounds can serve. The particular advantage of the method according to the invention is that (1) the silica coating avoids intolerance and rejection reactions by the recipient organism and (2) no damage to the matrices (collagen) by organic solvents can occur (the starting substrates are water-soluble in contrast) to the organic silicon compounds to be used according to the prior art such as TEOS).

Claims

claims

1. A process for the synthesis of amorphous silicon dioxide and other polymeric metal (IV) compounds, wherein (1) a template with (2) non-organic silicon compounds or metal (IV) compounds and / or aminosilanes and silazanes as substrate and (3) a polypeptide or a metal complex of a polypeptide is brought into contact for synthesis, the polypeptide comprising an animal, plant, bacterial or fungal carbonic anhydrase domain which has at least 25% sequence identity to the sequence shown in SEQ ID No. 1, or the polypeptide comprises an animal, bacterial, vegetable or fungus silicatein α-domain or silicatein β-domain which has at least 25% sequence identity to the sequence shown in SEQ ID No. 3 or in SEQ ID No. 5 ,

2. The method according to claim 1, characterized in that a template is used for the synthesis, which has functional groups with orthosilicic acid, oligomeric or polymeric silicas and their salts or other purely non-organic metal (IV) compounds or aminosilanes or silaza - interact.

3. The method according to claim 1, characterized in that compounds such as orthosilicic acid, oligomeric or polymeric silicas and their salts or other metal (IV) compounds are used as the substrate for the synthesis.

4. The method according to claim 1, characterized in that for synthesis one or more Si-N bonds containing aminosilanes or silazanes are used as the substrate.

5. The method according to any one of claims 1 to 4, wherein the templates are hydroxyl-containing molecules, molecular aggregates or surfaces.

6. The method according to claim 5, wherein the hydroxyl-containing molecules are collagen and / or silicatein.

7. The method of claim 6, wherein the collagen is a sponge collagen.

8. The method according to claim 7, wherein the collagen is a collagen from a sponge according to SEQ ID No. 7 or a homologous polypeptide which has at least 25% sequence identity in its amino acid sequence to the sequence shown in SEQ ID No. 7 or parts of it.

9. The method according to claim 6, wherein the silicatein is a silicatein from a sponge according to SEQ ID No. 3 or a homologous polypeptide which in its amino acid sequence has at least 25% sequence identity to that in SEQ ID No. 3 or in SEQ ID No. 5 shown sequence or parts thereof.

10.The method according to any one of claims 1 to 9, wherein a mixture of one or more templates is used.

11.The method according to any one of claims 1 to 10, wherein a polypeptide of a silicase from Suberites domuncula according to SEQ ID No. 1 or a homologous polypeptide which in the amino acid sequence of the carbonic anhydrase domain at least 25% sequence identity to that in SEQ ID No. 1 shown sequence, a metal complex of the polypeptide, or parts thereof is used.

12. The method according to any one of claims 1 to 11, wherein the polypeptide of a silicase from Suberites domuncula according to SEQ ID No. 1 or a homologous polypeptide which in the amino acid sequence of the carbonic anhydrase domain at least 25% sequence identity to that in SEQ ID No. 1 sequence has been provided in vivo, in a cell extract or lysate or in purified form.

13. The method according to claim 7, wherein the collagen from a sponge according to SEQ ID No. 7 or a homologous polypeptide which has in its amino acid sequence at least 25% sequence identity to the sequence shown in SEQ ID No. 7 or parts thereof in vivo, is provided in a cell extract or lysate or in a purified form.

14. The method according to any one of claims 1 to 13, wherein the surfaces are modified glass, metals, metal oxides, plastics, biopolymers or other materials.

15. The method according to any one of claims 1 to 13, wherein defined two- and three-dimensional structures are synthesized from amorphous silicon dioxide or other polymeric metal (IV) compounds.