Decomposition and modification of silicate and silicone by silicase and use of the reversible enzyme

Abstract

Silicatein is an enzyme of silicate-forming organisms used for the synthesis of their silicate scaffold. The present invention relates to the use of highly-expressed and highly active recombinant silicatein, silicatein isolated from natural sources after gene induction as well as silicatein-fusion proteins for the synthesis of amorphous silicon dioxide (silicic acids and silicates), siloxanes as well as modification of these compounds and their technical use.

1 . Method for the in vitro or in vivo degradation of amorphous or crystalline silicone dioxide (condensation products of the silicic acid, silicates), silicones and other silicon (IV)- or metal (IV)-compounds as well as of mixed polymers of these compounds, wherein a polypeptide or a metal complex of a polypeptide is used for the degradation, characterized in that the polypeptide comprises an animal, bacterial, plant or fungal carbonic anhydrase domain that exhibits a sequence similarity of at least 25% to the sequence shown in SEQ ID No. 1.  
     
     
         2 . Method for the synthesis of amorphous silicone dioxide (condensation products of the silicic acid, silicates), silicones and other silicon (IV)- or metal (IV)-compounds as well as of mixed polymers of these compounds, wherein a polypeptide or a metal complex of a polypeptide is used for the synthesis, characterized in that the polypeptide comprises an animal, bacterial, plant or fungal carbonic anhydrase domain that exhibits a sequence similarity of at least 25% to the sequence shown in SEQ ID No. 1.  
     
     
         3 . Method according to  claim 2 , characterized in that compounds such as silicic acids, monoalkoxysilantrioles, dialkoxysilandioles, trialkoxysilanoles, tetraalkoxysilanes, alkyl- or aryl-silantrioles, alkyl- or aryl-monoalkoxysilandioles, alkyl- or aryl-dialkoxysilanoles, alkyl- or aryl-trialkoxysilanes or other metal(IV)-compounds are used as reactants (substrates) for the synthesis.  
     
     
         4 . Method according to  claim 3 , wherein mixed polymers having a defined composition are produced by using defined mixtures of the compounds.  
     
     
         5 . Method according to any of  claims 2  to  4 , wherein the formation of defined two- and three-dimensional structures occurs by the polypeptide or a metal complex of the polypeptide or the binding of the polypeptide or a metal complexes of the polypeptide to other molecules or the surfaces of glass, metals, metal oxides, plastics, biopolymers or other materials as a template.  
     
     
         6 . Method for the modification of a silicic acid or silicon(IV)- or metal (IV)-compound containing structure or surface, wherein a polypeptide or a metal complex of a polypeptide is used for the modification, characterized in that the polypeptide comprises an animal, bacterial, plant or fungal carbonic anhydrase domain that exhibits a sequence similarity of at least 25% to the sequence shown in SEQ ID No. 1.  
     
     
         7 . Method according to  claim 6 , wherein the silicic acid-containing structure or surface is present in form of a precious stone or semi-precious stone.  
     
     
         8 . Method according to  claim 6  or  7 , wherein the modification comprises a smoothing, an etching or the production of burrows of the silicic acid or silicon(IV)- or metal(IV)-compound-containing structure or surface by the polypeptide or a metal complex of the polypeptide.  
     
     
         9 . Chemical compound or silicic acid-containing structure or surface, obtained according to a method of the preceding claims.  
     
     
         10 . Silicic acid-containing structure or surface according to  claim 9  in the form of a precious stone or semi-precious stone.  
     
     
         11 . Polypeptide of a silicase from Suberites domuncula according to SEQ ID Nr. 1 or a polypeptide being homologous thereto, which in the amino acid sequence of the carbonic anhydrase domain exhibits a sequence similarity of at least 25% to the sequence shown in SEQ ID No. 1, a metal complex of the polypeptide, or parts thereof.  
     
     
         12 . Nucleic acid, in particular according to SEQ ID No. 2, characterized in that it essentially encodes for a polypeptide according to  claim 11 .  
     
     
         13 . Nucleic acid according to  claim 12 , characterized in that it is present in the form of a DNA, cDNA, RNA or mixtures thereof.  
     
     
         14 . Nucleic acid according to  claim 12  or  13 , characterized in that the sequence of the nucleic acid has at least one intron and/or a polyA-sequence.  
     
     
         15 . Nucleic acid according to any of  claims 12  to  14  in the form of its complementary “antisense”-sequence.  
     
     
         16 . Nucleic acid according to any of  claims 12  to  15  in the form of a (a) fusion protein-(chimeric protein) construct, (b) construct having a separate protein-expression (protease-cleavage site) or (c) construct having a separate protein-expression (cassette-expression).  
     
     
         17 . Nucleic acid according to any of  claims 12  to  16 , characterized in that the nucleic acid has been synthetically produced.  
     
     
         18 . Vector, preferably in the form of a plasmid, shuttle vector, phagemid, cosmid, expression vector, retroviral vector, adenoviral vector or particle, nanoparticle or liposome, comprising a nucleic acid according to any of  claims 12  to  17 .  
     
     
         19 . Vector, preferably in the form of a nanoparticle or liposome, comprising a polypeptide according to  claim 11 .  
     
     
         20 . Host cell, transfected with a vector or infected or transduced with a particle according to  claim 18  or  19 .  
     
     
         21 . Host cell according to  claim 20 , characterized in that it expresses a polypeptide according to  claim 1 , a metal complex of the polypeptide or parts thereof.  
     
     
         22 . Polypeptide according to  claim 11 , characterized in that the polypeptide has been synthetically produced.  
     
     
         23 . Polypeptide according to  claim 11 , characterized in that the polypeptide or the metal complex of the polypeptide is present in a prokaryotic or eukaryotic cell extract or lysate.  
     
     
         24 . Polypeptide according to  claim 23 , characterized in that the polypeptide or the metal complex of the polypeptide is present being purified essentially free of other proteins.  
     
     
         25 . Method for identifying of inhibitors or activators of a polypeptide of a silicase from  Suberites domuncula  according to SEQ ID No. 1 or a polypeptide being homologous thereto that in the amino acid sequence of the carbonic anhydrase domain has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1, wherein a) a polypeptide of a silicase from  Suberites domuncula  according to SEQ ID No. 1 or a polypeptide being homologous thereto that in the amino acid sequence of the carbonic anhydrase domain has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1 is provided, b) the polypeptide from step a) is contacted with a potential inhibitor or activator, and c) the ability of the polypeptide is measured to degrade or synthesize silicate or silicones.  
     
     
         26 . Method according to  claim 25 , wherein the polypeptide of a silicase from  Suberites domuncula  according to SEQ ID No. 1 or a polypeptide being homologous thereto that in the amino acid sequence of the carbonic anhydrase domain has at least 25% sequence similarity to the sequence shown in SEQ ID No. 1 is provided in vivo, in a cellular extract or lysate or in purified form.  
     
     
         27 . Method for producing a pharmaceutical composition, comprising a) identifying of an inhibitor or activator according to  claim 25  or  26  and b) mixing of the identified inhibitor or activator with a pharmaceutically acceptable carrier or excipient.  
     
     
         28 . Use of a polypeptide or a nucleic acid or pharmaceutical composition according to any of the preceding claims for the prevention or therapy of silicosis.  
     
     
         29 . Use according to  claim 28 , wherein the prevention and therapy of silicosis occurs by dissolving of quartz crystals  
     
     
         30 . Use of a polypeptide or a nucleic acid or pharmaceutical composition according to any of the preceding claims for the resorption or for modulating the resorbability of silicones and silicone implants.  
     
     
         31 . Use of a nucleic acid according to any of the preceding claims for transfecting cells for the resorption or for modulating the resorbability of silicones and silicone implants.