BIOCATALYTICAL COMPOSITION

Abstract

The present invention relates to means and methods for protecting proteins and protein-type compounds in industrial and other applications. In particular, the invention provides a composition comprising at least one protein or protein-type compound immobilized at the surface of a solid carrier embedded in a protective material. Further, the present invention relates to methods for producing such a composition and to the use thereof in, for example, therapeutic applications. In particular, the system may be used to immobilize and protect enzymes on the surface of a carrier to generate a biocatalytical composition with increased resistance to various types of stresses.

Claims

1. A composition comprising: a solid carrier; at least one functional constituent selected from a protein and a protein-type compound, the at least one functional constituent immobilized on a surface of the solid carrier; and a protective layer for protecting the at least one functional constituent by completely embedding the at least one functional constituent in the protective layer, wherein the protective layer is built on the surface of the solid carrier with organo silane monomers as building blocks at least part of which are monomers capable of interacting with each other and the at least one functional constituent immobilized on the surface of the solid carrier and completely embedded in the protective layer so as to provide a first interaction of a weak force with the at least one functional constituent and a second interaction of form-locking the at least one functional constituent in the protective layer, wherein the first interaction and the second interaction are capable of retaining the at least one functional constituent in a form-locked manner that prevents release from the solid carrier, wherein the organo silane monomers comprise trivalent silane monomers and tetravalent silane monomers, wherein the at least one functional constituent selected from the protein or the protein-type compound is an enzyme or enzyme-type compound and wherein the enzyme or enzyme-type compound is covalently bound to the surface of the solid carrier, wherein the protective layer completely embeds a catalytically-active site of the enzyme or enzyme-type compound such that the enzyme or enzyme-type compound as immobilized and completely embedded in the protective layer retains activity of its catalytically-active site and its structural integrity.

2. The composition of claim 1, wherein the solid carrier is a nanoparticle.

3. The composition of claim 1, wherein the carrier is a particulate carrier with a particle size up to 100 m.

4. The composition according to claim 1, wherein the thickness of the protective layer is in a range selected from the group consisting of 1 nm to 100 nm, 1 nm to 50 nm, 1 nm to 30 nm, 1 nm to 25 nm, 1 nm to 20 nm, 1 nm to 15 nm, and 5 nm to 15 nm.

5. The composition according to claim 1, wherein the pore size of the protective layer is in a range selected from the group consisting of between 1 nm and 10 nm, between 2 nm and 9 nm, between 3 nm and 8 nm, between 4 nm and 7 nm, between 4 nm and 6 nm, and between 4 nm and 5 nm.

6. The composition of claim 1, wherein the pore size of the protective layer is dimensioned so as to allow for diffusion of molecules to the at least one functional constituent for interaction therewith during use of the composition.

7. The composition according to claim 1, further comprising at least one bi-functional cross-linker to bind the at least one functional constituent to the surface of the solid carrier, wherein the at least one functional constituent is selected from a protein and a protein-type compound.

8. The composition according to claim 1, wherein the first interaction between the monomer building blocks of the protective layer and the at least one functional constituent is effected between amino acid side chains of the protein or protein-type compound based on weak force interactions.

9. The composition according to claim 8, wherein a plurality of different building blocks are provided so that different building blocks interact with different functional constituents or different amino acid side chains.

10. The composition according to claim 8, wherein the at least one functional constituent of the protective layer interacting with the protein or protein-type compound is one of an alcohol, an amine, a carboxylate, an aromatic function, a thiol, a thioether, a guanidinium, an imidazole, an aliphatic chain, an amide, or a phenol.

11. The composition according to claim 8, wherein the at least one functional constituent selected from the protein or protein-type compound is an enzyme or enzyme-type compound selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

12. The composition of claim 1, wherein the solid carrier is a nanoparticle selected from the group consisting of organic nanoparticle, inorganic nanoparticle, organic-inorganic composite nanoparticle, self-assembling organic nanoparticle, mesoporous silica nanoparticle (SNP), gold nanoparticle, and titanium nanoparticle.

13. The composition of claim 1, wherein the carrier is a particulate carrier with a particle size in a range selected from the group consisting of between 20 nm and 1000 nm, between 200 nm and 500 nm, and between 300 nm and 400 nm.

14. The composition according to claim 7, wherein the at least one bi-functional cross-linker is a cross-linker for cross-linking amine to sulfhydryl (thiol) functions or a cross-linker for cross-linking sulhydryl to sulfhydryl (thiol) functions.

15. The composition according to claim 7, wherein the at least one bi-functional cross-linker is selected from the group consisting of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl] suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydrils, suflhydryl-reactive (2-pyridyldithio), BSOCOES (Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, DSP (Dithio-bis[succinimidyl propionate]), DTSSP (3,3-Dithiobis[sulfosuccinimidylpropionate]), DTBP (Dimethyl 3,3-dithiobispropionimidate.Math.2 HCl), DST (Disuccinimidyl tartarate), Sulfo-LC-SMPT (4-Sulfosuccinimidyl-6-[-methyl--(2-pyridyldithio)toluamido]hexanoate), SPDP (N-Succinimidyl 3-(2-pyridyldithio)-propionate), LC-SPDP (Succinimidyl 6-(3-[2-pyridyldithio]-propionamido)hexanoate), SMPT (4-Succinimidyloxycarbonyl-methyl-a-[2-pyridyldithio]toluene), DPDPB (1,4-Di-[3-(2-pyridyldithio)propionamido]butane), DTME (Dithio-bismaleimidoethane), and BMDB (1,4 bismaleimidyl-2,3-dihydroxybutane).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0190] FIG. 1A shows a schematic view of the process for the production of a protected enzyme on a solid carrier material; wherein

[0191] a: the enzyme is bound on a solid carrier material;

[0192] b: a protecting layer grows around the immobilized catalyst; and

[0193] c: over time the protecting layer can completely surround the enzyme.

[0194] FIG. 1B shows a schematic representation of the enzyme protection strategy; wherein

[0195] a: enzyme (circular shape) immobilization on the solid silica support (black);

[0196] b: self-assembly of the protection layer building blocks around the enzyme; and

[0197] c and d: protection layer growth (grey).

[0198] FIG. 2A shows a SEM micrograph of the silica nanoparticles (SNPs) used as carrier material.

[0199] FIG. 2B shows SEM micrographs of the enzyme-immobilized SNPs after 4 (left), 6 (middle), and 20 (right) hours of protective layer growth.

[0200] FIG. 3 shows a protective layer thickness measured after 4, 6, and 20 hours of reaction.

[0201] FIG. 4 shows relative activities of free (black squares) and protected (white squares) enzymes heat-stressed at 42 C. for increasing periods of time (activity values are normalized with the initial activity values measured without heat stress).

[0202] FIG. 5 shows relative activities of free (black squares) and protected (white squares) enzymes measured at increasing reaction temperatures.

[0203] FIG. 6 shows relative activities of free (white bars) and protected (black bars) enzymes incubated at different pH values and measured at pH 6.5 (activity values are normalized with the initial activity values).

[0204] FIG. 7 shows activities of free (empty squares) and protected (full squares) enzymes measured at different pH values (activity values are normalized with the maximum activity observed at pH 6.5).

[0205] FIG. 8 shows relative enzyme activity during the silane layer growth (activity values are normalized with the activity of the immobilized catalyst in the absence of protective layer).

[0206] FIG. 9 shows relative enzyme activity of the protected (black squares) and reference free enzymes (empty squares) at increasing heat stress durations (65 C.) (activity values are normalized with the activity of the immobilized catalyst before temperature stress).

[0207] FIG. 10 shows protective layer thickness measured at increasing reaction times for another example according to the invention.

[0208] FIG. 11 shows relative activities of free lactase and lactase protected with a layer made of APTES-TEOS mixture or with a layer made of silane mixture heat-stressed at 50 C. for one hour.

DETAILED DESCRIPTION

[0209] According to FIG. 1A, showing a schematic view of the process for the production of a protected enzyme on a solid carrier material, the enzyme is first bound on a solid carrier material (compare step a)). Then, a protecting layer grows around the immobilized catalyst (compare step b)). Thereafter, over-time the protecting layer can completely surround the enzyme (compare step c)).

[0210] Then, FIG. 1B shows a schematic representation of the enzyme protection strategy. First, the enzyme (circular shape) immobilization on the solid silica support (black, compare step a).

[0211] Then, self-assembly of the protection layer building blocks around the enzyme takes place. Then, compare steps c and d, the protection layer grows (grey). The environment around the enzyme (i.e., interactions between the enzyme outer surface and the cavities formed in the organosilica layer) provide a great conformational stabilization effect. In this example, the carrier material is silica (nanoparticle), and the protecting layer is organosilica (polysilsesquioxane) produced by the polycondensation reaction of silica precursors (tetraorthosilicate and organo-silanes).

[0212] It is noted that the increased stabilization of the tertiary protein structure against stress achieved is concluded from activity measurements as only a properly folded enzyme is likely to be active.

[0213] In FIG. 2A, SEM micrographs of the silica nanoparticles (SNPs) used as carrier material are shown, and SEM micrographs of the enzyme-immobilized SNPs after 4 (left), 6 (middle), and 20 (right) hours of protective layer growth are shown in FIG. 2B.

EXAMPLES

Example 1: Lactase Immobilization on a Solid Carrier Material and Protection by an Organo-Silica (i.e., Silsesquioxane) Layer

[0214] Lactase/-galactosidase (EC 3.2.1.23) immobilization on a solid carrier material such as silica nanoparticles (SNPs) and protection involves four main steps that are: [0215] i. Surface modification of the SNPs in order to introduce anchoring points (i e, amine) for the further chemical coupling with the enzyme; [0216] ii. Chemical reaction of the introduced amine moieties with a bi-functional cross-linker (e.g., glutaraldehyde); [0217] iii. Enzyme coupling at the surface of the SNPs through the free active functions of the bi-functional cross-linker; and [0218] iv. Polycondensation of silane building-blocks around both immobilized enzymes and free surface of the SNPs to yield a protective layer.

[0219] This synthetic procedure allows producing a protective layer at the surface of the SNPs surrounding and thus protecting the enzyme. The thickness of the produced protective layer can be adjusted by design, depending on the targeted application. [0220] i) SNPs were produced using the conventional Stber method adapted from the report of Imhof et al. (J. Phys. Chem. B 1999, 103, 1408), as follows. Ethanol (345.4 ml), ammonia 25% (39.3 ml) and TEOS (tetraethylorthosilicate, 15.3 ml) were mixed in a round bottom flask and this mixture was stirred at 600 rpm during 20 hours, at a constant temperature of 20 C. The resulting precipitate was consequently washed twice with ethanol and twice with water, and freeze-dried to yield bare SNPs that were characterized using scanning electron microscopy (Zeiss, SUPRA 40 VP). The acquired micrographs were used for particle size measurement using the Analysis (Olympus) software package (statistical analysis carried out on 100 measurements). In FIG. 2A, there is given a representative micrograph of the produced SNPs. [0221] ii) In order to introduce, at the surface of the SNPs, amine functions that allowed the further anchoring of the to-be-protected enzyme, they were reacted with an aminosilane. It is important to note that this modification should be only partial so as to leave silanol groups for the further attachment of the protective layer. [0222] In more details, SNPs in suspension in water (18 mL; 3.2 mg/mi) were incubated with APTES (3-aminopropyltriethoxysilane, 11 mg) during 90 minutes at 20 C. After two washing steps in water, the resulting amino-modified SNPs were reacted during 30 minutes with a bi-functional cross-linker (to allow the further immobilization of the enzyme), glutaraldehyde, at a final concentration of 1 g/L. [0223] iii) After two washing steps in water, the resulting SNPs were re-suspended in a MES (2-(N-morpholino) ethanesulfonic acid) buffer (pH 6.2, 1 mM, 5 mM MgCl.sub.2) and incubated for 1 hour at 20 C. with the enzyme, Lactase, (100 g/ml) under magnetic stirring at 400 rpm. [0224] iv) The protection of the enzyme immobilized on SNPs was carried out by incubating the produced enzyme-immobilized SNPs with a mixture of silane building blocks that self-assembled around the enzyme and underwent a polycondensation reaction that created a protecting layer around the enzyme. Adequate building blocks have to be selected in dependence from the protein or protein-type compound of interest and its amino acid residues on the surface. Examples for a preferred selection of self-assembling building blocks are given in TABLE 1, which provides a list of putative protective layer building blocks and the main forces, which interact between the protein surface's amino acid residues of the protected protein and the self-assembled building block. The polycondensation reaction also occurred at the bare surface of the SNPs allowing the attachment of this layer at the surface of the SNPs. To that end, enzyme-immobilized SNPs (18 mL; 3.2 mg/mi) were first reacted at 20 C. under stirring at 400 rpm with 36 l of TEOS. After 2 hours of reaction, 18 l of APTES were added and the protective layer was allowed to grow over time at 4 C. Samples of SNPs were collected every 2 hours and the reaction was stopped after 20 hours by two washing steps in MES buffer. The protective silane layer thickness at different time points was measured as described above and is reported in FIG. 2B and FIG. 3. From these results, it could be seen that the organosilane layer is 2, 10, and 25 nm thick after respectively 4, 6, and 20 hours of reaction. These results confirmed the possibility to control the organosilica layer growth at the surface of enzyme-immobilized SNPs.

[0225] The enzymatic activity of the so-produced particles was assayed using ortho-nitrophenyl--galactoside (ONPG) as artificial substrate and following spectrophotometric ally the appearance of the product ortho-nitrophenol (ONP) at 420 nm revealed in alkaline conditions. In more details, SNPs were collected at increasing durations of silane polycondensation and washed twice in MES buffer. To measure the lactase activity, SNPs were incubated for 5 minutes at 40 C. with ONPG (40 mM) at pH 6.5 and the reaction was stopped by the addition of an equal volume of an aqueous solution of Na.sub.2CO.sub.3 (1 M). The result showed that 45% of the initial enzymatic activity was present on the particles possessing a protective layer of 25 nm confirming that even when the enzyme is buried into an organosilica protective layer, it maintains partially its activity.

[0226] With respect to FIG. 3, it is noted that the layer thickness obtained over time depends on the kinetics of the polycondensation reaction and correspondingly will depend on the (i) time, (ii) temperature and (iii) mixture of organosilanes used. For (i) it is clear that the longer the reaction is kept, the thicker the protective layer will be. For the temperature, it will be understood that the higher the temperature, the faster the kinetics and therefore the thicker the protective layer (and vice versa). With respect to the mixture of monomers, it is noted that the presence of organosilanes with basic character (APTES or UPTES) will boost the protective layer growth.

[0227] In FIG. 3, an initial delay can be observed which currently is attributed to an initial pre-hydrolysis and solubilization of the more hydrophobic monomers. Once this initial reactions are done, the thickness of the protective layer becomes measurable (e.g., by scanning electron micrographs and statistical analysis of the particle size with a software). If the protective layer has reached the thickness needed and the reaction and thus further thickening of the protective layer shall be stopped, this can be done in case of a protective layer building polycondensation reaction by washing the particles and removing the unreacted monomers.

TABLE-US-00001 TABLE 1 List of protection layer building blocks and the main forces interacting between these building blocks and the amino acid residues on the surface of the protein or protein-type compound of interest, which is embedded in the protective layer. The selection of the adequate building blocks should be adapted to the present protein surface amino acid residues. Protection layer building Protein surface amino Main interactions block acids (3-letter-code) involved Benzyltriethoxysilane Phe, Tyr, Trp p-p (aromatic) interactions Propyltrimethoxysilane Gly, Ala, Leu, Ile, Val, Pro van der Waals Isobutyltriethoxysilane Gly, Ala, Leu, Ile, Val, Pro van der Waals n-Octyltriethoxysilane Gly, Ala, Leu, Ile, Val, Pro van der Waals Hydroxymethyltriethoxysilane Ser, Thr, Asp, Glu, Asn, H-bonding Gln, Tyr Bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane Ser, Thr, Asp, Glu, Asn, H-bonding Gln, Tyr Aminopropyltriethoxysilane Asp, Glu Ionic Tetraorthosilicate Lys, Arg, His Ionic Carboxyethylsilanetriol Lys, Arg, His Ionic

[0228] With respect to Table 1, the following is noted: First, while Table 1 is given in connection with the embedding of a specific functional constituent, it will be understood that the information disclosed by Table 1 and in connection therewith will be relevant to other functional constituents as well. Then, it will be understood by the average skilled person that the list of Table 1 is not exhaustive and that there are other organosilane monomers that can be used for the method according to the invention.

[0229] In this context, it is further noted that the list is not exhaustive as it would be difficult to establish an exhaustive one, e.g., as non-commercial silanes could also be produced and used. Furthermore, on certain occasions, it may be advantageous to use organosilanes carrying large and bulky groups, e.g., octadecyltrimethoxysilane and triphenyl-triethoxysilane, e.g., to obtain sufficiently large pores.

[0230] Additionally, it will be noticed that the silanes in the table as well as frequently mentioned throughout other parts of the disclosure are given as triethoxy derivatives; yet, referring to a single derivative rather than all possible derivatives such as, e.g., tri-methoxy or tri-hydroxyethoxy derivatives has been done to simplify reading and to simultaneously direct the reader to organo silanes readily available, not in order to restrict the scope of the disclosure. The average skilled person will understand that reference could have been made to silanes in general (e.g., aminopropyl silane instead of referring to the aminopropyltriethoxy silane in the table. Furthermore, the list is not even complete with respect to silanes particularly relevant for specific main interactions. As an example, Ureidopropyltriethoxysilane and (N-Acetylglycyl)-3-aminopropyltrimethoxysilane, could be included as further strong H-bonding donor acceptor monomers.

[0231] Then, it is noted that CYS and MET are not listed in Table 1. However, if these are present at the surface of the functional constituent, it is possible to select appropriate organosilanes that interact with these amino acids. For example, use can be made of the fact that these amino acids can form covalent disulfide bridges (SS) to suitable organosilanes bearing an SH group, such as (3-Mercaptopropyl)trimethoxysilane or, in a more generic way (3-Mercaptopropyl) silane Hence, e.g. organo silanes bearing a functional SH group could also be added to the list.

[0232] Finally, it will be obvious that Table 1 will not only be referred to with respect to encapsulation of Lactase immobilization but will be found instructive by the average skilled person intending to embed other functional constituents as well,

Example 2: Protection Against Thermal Stress

[0233] The thermal resistance of enzymes protected with the method described herein was tested using lactase-modified particles with a protective layer of 20 nm, produced as described in Example 1. The catalytic activities were measured using the ONPG colorimetric method also described in example 1.

[0234] First the protected and free enzymes were thermally stressed at 42 C. for increasing periods of time, and the activity measured; cf. FIG. 4.

[0235] It could be seen that while the activity of the free enzymes dropped down to 70% after 5 min, 45% after 30 min and 8% after 60 minutes; the protected enzyme remained for all the tested conditions at activity values higher than 90%. Interestingly, the activity even increased to 112% for 20 min and 30 min of heat stress. This set of results clearly demonstrated the advantage of the enzyme protection strategy described herein.

[0236] In addition, the enzyme activity was measured at increasing temperature values; the results are reported in FIG. 5.

[0237] From the results reported in FIG. 5, it could be seen than the activity of the free enzyme dropped down to a value of 52% at 50 C., and no activity could be measured at 50, 55 and 60 C. For the protected enzyme, interestingly, the activity increased to 106%, 113% and 111% for reaction temperatures of 45 C., 50 C. and 55 C. A slight decrease of 11% is observed for the reaction measured at 60 C.

Example 3: pH Resistance and pH Range Broadening of Protected Lactases

[0238] The resistance of enzymes protected with the method described herein and the broadening of their pH activity range was tested using lactase-modified particles with a protective layer of 20 nm; produced as described in example 1. The catalytic activities were measured using the ONPG colorimetric method also described in example 1.

[0239] First, free and immobilized enzymes were incubated during 15 minutes at different pH values (4.8, 6.5, 7.6, 8.8); the pH value was then adjusted to the optimal catalytic pH (6.5) and the activity of the different systems measured; the results are reported in FIG. 6. It could be seen that while the treatment at pH 6.5 did affect neither the immobilized enzyme nor its free counterpart, a treatment at pH 4.8 caused a drastic decrease in activity of the free enzyme down to 25%, while the protected enzyme remained unaffected. At pH values of 7.6 and 8.8, the free enzyme lost 15% and 28% of activity respectively while the protected enzyme was unaffected at pH 7.6 and lost only 10% of activity at pH 8.8.

[0240] Additionally, catalytic activities of free and immobilized lactases were measured using the ONPG colorimetric method described in example 1 at different pH values (5.5, 6.0, 6.5, 7.5 and 8.0). The relative activity values measured are reported in FIG. 7.

[0241] Both enzymatic systems had an optimal pH value of 6.5. Increasing the pH to 7.5 and 8.0, the free enzyme showed a decay in activity of 20% and 40% respectively, while the protected enzyme lost only 5% and 18% in the same conditions. For acidic pH values, the free enzyme lost 40% and 80% of activity for pH value of 6.0 and 5.5, respectively; while the protected enzyme lost only 2% and 15%. Those results confirmed the protection of the enzyme resulting in the broadening of its activity range.

Example 4: Protection Against Protease Attack

[0242] The resistance to proteases of enzymes protected with the method described herein was tested using lactase-modified particles with a protective layer of 20 nm, produced as described in example 1. Free and protected enzymes were incubated with proteinase K and trypsin (1 mg/mL) for 60 min at 37 C. in 0.1 M Tris-HCl (pH 7.4). While the activity of the free enzyme dropped down to zero, the activity of the protected enzyme remained unchanged.

Example 5: Acid Phosphatase Immobilization on SNPs and Protection by an Organosilica (i.e. Silsesquioxane) Layer, and Temperature Stress Test

[0243] Acid phosphatase (EC 3.1.3.2) immobilization on SNPs and protection by growing a layer of organosilanes have been performed as described in example 1. Protected catalysts, with increasing protection layer thicknesses, were produced and assayed using para-nitrophenylphosphate (pNPP) as artificial substrate. The appearance of the product p-nitrophenol (pNP) at 405 nm was followed spectrophotometrically and revealed in alkaline conditions.

[0244] Briefly, to measure the acid phosphatase activity, the protected biocatalysts were incubated for 5 minutes at 37 C. with pNPP (15 mM) at pH 4.8 and the reaction was stopped by the addition of an equal volume of an aqueous solution of NaOH (100 mM); the results are given in FIG. 8. It is shown that the enzymatic activity does increase with the presence of the protective layer.

[0245] The resistance to temperature was assayed by incubating the produced particles (and soluble reference enzyme) at 65 C. for increasing durations. The results of activity are reported in FIG. 9. It is demonstrated that while the free reference enzyme lost more than 90% of activity after 10 minutes and more than 95% after 30 minutes, the protected enzyme maintains as much as 80% after 10 minutes of treatment and more than 75% after 60 minutes.

Example 6: Lactase Immobilization on SNPs and Protection by a Layer Made of a Silane Mixture

[0246] The Lactase/-galactosidase (EC 3.2.1.23) immobilization on SNPs has been performed as described in example 1. The protection of the enzyme immobilized on SNPs was carried out by incubating the produced enzyme-immobilized SNPs with a mixture of silane building blocks that self-assembled around the enzyme and underwent a polycondensation reaction that created a protecting layer around the enzyme. The used silanes were: APTES, TEOS, benzyltriethoxysilane (BTES), Propyltrimethoxysilane (PTES), and Hydroxymethyltriethoxysilane (HMTES). In more details, enzyme-immobilized SNPs (18 mL; 3.2 mg/mi) were first reacted at 20 C. under stirring at 400 rpm with 36 l of TEOS. After 1 hour of reaction, 18 l of APTES, 18 l BTES, 18 l PTES and 36 l HMTES were added and the protective layer was allowed to grow at 20 C. Samples of SNPs were collected at increasing reaction times and the reaction was stopped after 20 hours by two washing steps in MES buffer. The protective silane layer thickness, at different time points, was measured as previously described. As shown in FIG. 10, the organosilane layer was 2, 8, 12, and 16 nm thick after 4, 6, 10, 17, and 20 hours of reaction, respectively.

[0247] The thermal resistance of the so-produced protected lactase was tested by incubation at for 60 min and compared to catalyst protected using a mixture of APTES-TEOS as shown in FIG. 5. The result showed that while the activity of the free lactase was lower than 5% after 1 hour treatment at 50 C., the activity of the lactase protected with a layer made of APTES-TEOS or made of silane mixture was higher than 110% and 150% respectively (FIG. 11).

[0248] It is noted that the present invention claims priority of EP 13 17 850.4. The priority giving document is fully enclosed herein for purposes of disclosure.

[0249] Accordingly, what has been described above inter alia is a composition comprising at least one protein or protein-type compound and optionally further comprising at least one molecule selected from the groups of adaptor molecules, anchoring molecules, scaffold molecules and/or receptor molecules, immobilized at the surface of a solid carrier, wherein the protein or protein-type compound and the at least one optional molecule is fully or partially embedded in a protective material comprised of self-assembling building blocks, which building blocks comprise functional groups, which interact with the chemical groups of the protein or protein-type compound and the at least one optional molecule such that a porous nano-environment is established on the carrier surface and around the immobilized protein or protein-type compound and the at least one optional molecule which stabilizes the native conformation and preserves the function of the protein or protein-type compound and the at least one optional molecule.

[0250] Furthermore, it has been suggested that in such a composition the solid carrier is a nanoparticle, particularly a silica nanoparticle (SNP), particularly a gold nanoparticle, particularly a titanium nanoparticle.

[0251] Furthermore, it has been suggested that in such a composition the binding of the protein or protein-type compound to the surface of the solid carrier is covalent binding.

[0252] Furthermore, it has been suggested that in such a composition the size of the nanoparticle is in a range of between 20 nm and 1000 nm, particularly of between 200 and 500 nm., particularly between 300 and 400 nm.

[0253] Furthermore, it has been suggested that in such a composition the thickness of the protective material ranges from 1 nm to 100 nm, 1 nm to 50 nm, 1 nm to 30 nm, 1 nm to 25 nm, 1 nm to 20 nm, 1 nm to 15 nm, preferably 5 nm to 15 nm.

[0254] Furthermore, it has been suggested that in such a composition the self-assembled protective material has a pore size which allows the diffusion of substrates, particularly a pore size of between 1 nm and 10 nm, particularly between 2 nm and 9 nm, particularly between 3 nm and 8 nm, particularly between 4 nm and 7 nm, particularly between 4 nm and 6 nm, particularly between 4 nm and 5 nm.

[0255] Furthermore, it has been suggested that in such a composition the functional groups of the self-assembling protective material are groups interacting with the amino acid side chains of the protein or protein-type compound, particularly based on weak force interactions.

[0256] Furthermore, it has been suggested that in such composition the protective material is organosilica.

[0257] Furthermore, it has been suggested that the protein or protein-type compound is an enzyme or enzyme-type compound, particularly an enzyme or enzyme-type compound, which is selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerises and/or ligases.

[0258] Furthermore, it has been suggested that in such a composition the protein or protein-type compound and/or at least one of the optional molecules is bound to the surface of the solid carrier by a bi-functional cross-linker, particularly a bi-functional cross-linker selected from the group of glutaraldehyde, disuccinimidyl tartrate, bis[sulfosuccinimidyl] suberate, ethylene glycolbis(sulfosuccinimidylsuccinate), dimethyl adipimidate, dimethyl pimelimidate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, 1,5-difluoro-2,4-dinitrobenzene, activated sulfhydryls (e.g. sulfhydryl-reactive 2-pyridyldithio).

[0259] Furthermore, it has been suggested that in such a composition the protective material provides protection to: [0260] a) a pH, different from the optimal pH of the at least one protein or protein-type compound, wherein the pH value differs from the optimal pH by a value of +/5, +/4; +/3, +/2, +/1, +/0.5 pH units; and/or [0261] b) chemical stresses; and/or [0262] c) biological stresses; and/or [0263] d) solvents; and/or [0264] e) physical stress; and/or [0265] f) elevated temperatures, which exceed the optimal temperature for the unprotected protein or protein-type compound by 60 C., particularly by 50 C., particularly by 40 C., particularly by 30 C., particularly by 20 C., particularly by 10 C., particularly by 5 C.; and/or [0266] g) reduced temperatures, which deviate from the optimal temperature for the unprotected protein or protein-type compound by 60 C., particularly by 50 C., particularly by particularly by 30 C., particularly by 20 C., particularly by 10 C., particularly by 5 C.

[0267] Furthermore, it has been suggested that in such a composition the immobilized and protected enzyme has: [0268] a) an increased activity under stress conditions when compared to the free-unprotected enzyme; and/or [0269] b) an increased recoverability for use in continuous operation compared to the free-unprotected enzyme.

[0270] Then, a method for producing such a composition according to the invention is suggested comprising the steps of: [0271] a) obtaining a solid carrier; [0272] b) immobilizing at least one protein or protein-type compound of interest, particularly at least one enzyme or enzyme-type compound, and, optionally, at least one optional molecule at the surface of the carrier; [0273] c) incubating the at least one protein or protein-type compound and, the optional molecule bound at the surface of the solid carrier with self-assembling building-blocks to yield a porous nano-environment around the free surface of the solid carrier and the at least one protein and/or protein-type compound and optional molecule bound at the surface of the solid carrier, and [0274] d) stopping the self-assembly reaction of the protective material at a specific time point to obtain a preferred protective layer with a desired thickness.

[0275] Furthermore, it has been suggested that such a composition be used in a catalytic process.

[0276] Furthermore, it has been suggested that such a composition be used in therapy, for example, of sphingomyelinase deficiency (ASMD) syndrome, Niemann-Pick Disease (NPD), lysosomal storage diseases, Gaucher disease, Fabry disease, MPS I, MPS II, MPS VI and Glycogen storage disease type II, cancer, allergic diseases, metabolic diseases, cardiovascular diseases, autoimmune diseases, nervous system disease, lymphatic disease and viral disease.

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