WO2005028604A1

WO2005028604A1 - Silica derived sol-gels sensitive to water content change

Info

Publication number: WO2005028604A1
Application number: PCT/US2004/030990
Authority: WO
Inventors: Nathaniel Todd Becker; Dave C. Bakul; Kiranmayi Deshpande; Mark S. Gebert; Joseph C. Mcauliffe; Wyatt Charles Smith
Original assignee: Genencor International, Inc.; Southern Illinois University
Priority date: 2003-09-19
Filing date: 2004-09-17
Publication date: 2005-03-31

Abstract

The present invention provides an enzyme-containing sol-gel composition wherein one or more enzymes are stably entrapped within the gels and are released from the gels in responsive to an increase in environmental water percentage (by weight). The enzyme-containing sol-gels are prepared from starting materials comprising one or more aminoalkylsilane precursors such as bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane, and one or more alkoxysilane precursors such as dialkyldialkoxysilane and alkyltrialkoxysilane. The present invention further provides a liquid detergent, a liquid soap or a shampoo formulation comprising an enzyme-entrapped sol-gel system, wherein the enzymes are released upon dilution of the formulation in water. The silica gel matrices not only improve the thermal stability of enzymes but also prevent the enzyme denaturation due to proteolysis/autolysis or interference from other components of the formulation.

Description

SILICA DERIVED SOL-GELS SENSITIVE TO WATER CONTENT CHANGE

FIELD OF INVENTION The present invention relates to silica sol-gels having proteins such as enzymes entrapped, wherein the proteins are released when the sol-gels are placed in a solution having a high water percentage by weight. The present invention is useful in detergent and fabric care applications. The present invention is applicable to cleaning products such as liquid detergents, soaps, and shampoos.

BACKGROUND OF THE INVENTION The detergent industry has been actively advocating the use of the enzyme technology in detergent formulations. Proteases are used for protein-based stains such as blood, egg, milk and grass. They bring about hydrolysis of peptide linkages within protein molecules. α-Amylases, which hydro lyse α-1, 4- glycosidic bonds, have been used to break down stubborn starch based stains. Lipases, which hydrolyze ester bond linkage, are used to fight triglyceride based stains. Cellulases, the most recent introduction to the detergent industry, are used mainly for their antipilling and color revival properties. Use of enzymes in the detergent industry is now an accepted technology. The recent endeavors have been focused on developing methods to stabilize the protein structure of enzyme molecules in a detergent environment. Linear alkylbenzene sulfonates and other anionic surfactants that are commonly used in detergents have a tendency to unfold proteins, thereby deactivating them. Development of technology which stabilizes enzymes in a multiple component detergent environment has been a challenging task. A protein such as an enzyme is usually deactivated by denaturation, unfolding, catalytic site inactivation, or proteolysis. Denaturation is usually caused by thermal unfolding, or oxidation, or covalent modification of reactive groups. Maintenance of a certain temperature and pH range and absence of chemical denaturants in the detergent environment is found helpful in preventing the protein unfolding. Catalytic site deactivation can be prevented by maintaining a sufficient level of cofactors such as a metal ion. The rate of proteolysis has been lowered by using water sequestering agents such as sugars, sugar alcohols such as sorbitol, and polyols such as propylene glycol or polyethylene glycol. Proteolytic inhibitors, which include series of small molecular weight carboxylic acid salts such as formate, acetate, propionate and butyrate and boric acid, have also been used. For example, calcium salts are known to stabilize subtilisin type proteases and Bacillus α- amylases (Becker, et al, "Enzymes in detergency", Eds., VanEe, Misset, and Baas, 69: 299). Granulation techniques have been used to separate enzymes from each other and from the surfactants of the detergent. Polyethylene glycol and polyvinyl alcohol are useful in this regard. Other attempts to separate enzymes from detergent involved the formation of liquid emulsions. The present detergent technology is faced by many challenges. Zeolites, phosphonic acids, polyacrylic acids, and polycarboxylate polymer builders, which are used in detergent compositions, sequester calcium ions from the active sites of the enzymes thus deactivating them. Linear alkyl benzene sulfonates and alkyl sulfonates are amongst the most harmful chemicals for enzyme stability, followed by alkyl ethoxysulfonates. Reactive oxygen species are another problem. Few enzyme species are known yet to overcome the presence of chlorine bleach. Another challenge faced by the industry is development of dust-free enzyme granules for improved hygiene of workers involved in product manufacture. Stability of multi-enzyme formulations, preventing auto lysis of proteases and proteolysis of other enzymes represent additional hurdles. Studies indicate that depending on the matrix precursor and the protocol used, proteins with molecular weight of 8,000-15,000 can be irreversibly encapsulated in sol-gels (Gill, et al, J. Am. Chem. Soc, 120: 8587(1998); Shtelzer, et el, Biotechnol. Appl. Biochem., 15: 227(1992)). Absorbance and fluorescence studies of albumin, cytochrome c, cytochrome-c peroxidase, myoglobin, glucose oxidase, monelhn, and glutamate dehydrogenase encapsulated in silica gels have indicated that proteins are entrapped in their native conformations within rigid polymer cages, in which global movements such as unfolding, rotation are restricted but segmental motions including those required for substrate binding and catalysis are largely unaffected (Edmiston, et al, J. Colloid Intrface Sci., 163: 395(1994); Hartnett, et al, Anal. Chem., 71 : 1215(1999)). Considerable work has been directed towards integrating sol-gel chemistry and encapsulation systems with various applications. Activation of lipases on encapsulation in poly alkyl siloxanes (Anvir, et al, Biochemical Aspects of Sol-gel Science and Technology, 1996, Kluwer Academic publications; Reez, Adv. Mater., 9: 943(1997)), immobilization of variety of cells and proteins in glycerol esters of silicates and siloxanes (Gill, et al, J. Am. Chem. Soc, 120: 8587(1998)), establish the biochemical aspect of sol-gel science. Dave, et al. (Anal. Chem., 66: 22 (1994)) disclose that proteins trapped in a silica sol- gel matrix retain their biological function and can be used in optical biosensors. Rao, et al. (Sol-Gel Science and Technology, 26: 553-560(2003)) report that silica- based sol-gels exhibit bulk volume changes and active mechanical responses with respect to temperature, pH, salt, and solvents. U.S. Patent No. 6,495,352 discloses a method to encapsulate molecules, comprising: forming a silica sol from a solution of a silicon oxide and alkali metal oxide in water; adjusting the pH to a pH value less than approximately 7 to stabilize the silica sol, forming a silica sol matrix solution; adding a solution containing an organic compound to be encapsulated to form a silica sol matrix encapsulating said organic compound; aging said silica sol matrix encapsulating said organic compound; and forming a material selected from the group selected of a thin film and a gel. EP 0676 414 Bl discloses a process for the preparation of immobilized lipases, characterized in that lipases are entrapped in a hydrophobic silica matrix containing organic substituents attached through Si-C bonds. Santos, et. al. (Biomaterials, 20: 1695-1700 (1999)) report that silica xerogels containing trypsin inhibitor made from teframethylorthosilicate (TMOS) slowly release 20- 40% of the entrapped trypsin inhibitor in a diffusion controlled manner over nine weeks. U.S. Patent No. 6,756,217 (Dave, et al) discloses a porous glass composite material comprising (1) at least one alkoxodisilane precursor having the general formula (OR^])₃Si- spacer-Si(OR )₃, where R and R may be the same or different and may comprise hydrogen, alkyl, alkenyl, alkynyl, or aryl groups as defined herein below, and the two silicon atoms are bridged by a spacer unit comprising an organic unit, an inorganic unit, biological unit and combinations thereof; and (2) water. None of the references have disclosed a sol-gel formulation wherein the entrapped proteins are stable and their release from the gel is triggered when the formulation is placed in a solution having a high water percentage. There is a need for a stable protein formulation wherein the protein release can be triggered when the formulation is diluted in water. Such formulations are useful in cleaning products such as liquid detergents, soaps, and shampoos.

SUMMARY OF INVENTION The present invention provides an enzyme-containing sol-gel composition that is responsive to water percentage change. The present invention also provides a liquid detergent, a liquid soap or a shampoo formulation comprising enzyme-containing sol-gels. Enzyme-containing sol-gels are prepared from starting materials comprising one or more aminoalkylsilane precursors such as bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane; and one or more alkoxysilane precursors such as dialkyldialkoxysilane and alkyltrialkoxysilane, wherein one or more enzymes are contained within the sol-gels, and at least 30% of said one or more enzymes are released from the sol- gels within an hour upon diluting the sol-gels into a solution that increases the water percentage by weight to 86% or higher. The enzyme-containing sol-gels are prepared by the steps of (a) preparing starting materials comprising one or more aminoalkylsilane precursors selecting from the group consisting of bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane) amine, and aminoalkyltrialkoxysilane; and one or more alkoxysilane precursors selected from the group consisting of dialkyldialkoxysilane and alkyltrialkoxysilane; (b) adding to the starting materials a first aqueous solution to hydrolyze the silane precursors to silicate or organosiloxane sols; (c) adding to the sols a second aqueous solution containing one or more enzymes; and (d) incubating (c) to form gels; wherein the one or more enzymes are entrapped within the gels and are releasable when the gels are placed in a solution having a water percentage by weight of 86% or more. The present invention is based on use of organosiloxane sol-gels for the controlled release of proteins in detergent and fabric care applications. The present invention is applicable to cleaning products such as liquid detergents, soaps, and shampoos. The silica gel matrices not only improve the thermal stability of enzymes but also prevent the enzyme denaturation due to proteolysis/auto lysis or interference from other components of the cleaning products.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows that SP-enTMOS (2:3 moles) gels swell in water and shrink in TIDE*¹ detergent. Figure 2 shows that SP-ATMOS (2:4 moles) gels swell in water and shrink in TIDE^®detergent. Figure 3 shows the percentage of enzyme leaked from gels (Sp-enTMOS, ESP, ESF- NS and SP-ATMOS (-ve)) into detergent during static storage in detergent for 1, 2, 3, or 4 weeks. Figure 4 shows the percentage of enzyme released from gels (Sp-enTMOS, ESP, ESF-NS and SP-ATMOS (-ve)) upon a 10-fold dilution of gel-containing detergent with water within an hour; the gels had been stored in TIDE^® for one, two, three or four weeks. DETAILED DESCRIPTION OF THE INVENTION The present invention is based on use of organosiloxane sol-gels for the controlled release of proteins in detergent and fabric care applications. The present invention is applicable to cleaning products such as liquid detergents, powder detergent, soaps, shampoos, and fabric cleaning agents. The general strategy of the present invention is based on encapsulation of proteins in a porous silica sol-gel matrix, which does not release the proteins when the matrix is placed in a solution having low water content (percentage by weight) but releases the proteins when the solution is diluted with water. In one embodiment of the invention, the protein is an enzyme. In another embodiment of the invention, the solution is a liquid detergent such as a laundry detergent. In the present invention, the characteristics of sol-gels are used both for stabilizing the protein activity and for controlling the release of proteins. Proteins such as enzymes are encapsulated and stabilized in sol-gels. When the external environment of sol-gels changes to a higher water percentage by weight (when the sol-gel containing matrix is diluted with water), the enzymes are released from the gels. Silica sol-gels refer to silicon dioxide based materials made through a sol-gel process.

Sols are formed first, which consist of a colloidal solution of very small (nanometer sized) polysiloxane particles formed through hydrolysis of the silane starting materials. Further polymerization/chemical reaction/hydrolysis converts the sols into gels by chemically linking together the individual colloidal sol particles into monolithic gels. The sol-gel process involves low-temperature hydrolysis of suitable monomeric precursors and is highly suitable for microencapsulation of a variety of molecules such as enzymes that cannot withstand high temperatures. The sol is usually formed by hydrolysis of an alkoxy silane precursor followed by condensation to yield a polymeric oxo-bridged SiO₂ network. In the process, molecules of the corresponding alcohol are liberated. A sol can also be formed by the neutralization of an alkali metal salt of a silicate or organosiliconate with an acid. The initial hydrolysis and polycondensation reactions in a localized region lead to formation of colloidal particles. A suspension containing these colloidal particles is called a sol. As the interconnection between these particles increases, the viscosity of the sol starts to increase and leads to the formation of a solid gel. Although the nature of individual events is somewhat random and the geometry and pore-size distribution of the product gel are difficult to control, the nature of the final polymeric gel can be regulated to a certain extent by controlling the rates of the individual steps. The protein to be encapsulated is added to the sol after partial hydrolysis or neutralization of the precursor. As the degree of cross-linking from polycondensation increases, the gel becomes viscous and solidifies. Protons or hydroxide ions are required for catalysis in silica sol-gel formation; therefore, the pH of the reaction medium is an important factor that affects the stoichiomerry of the final gel. Both acid and base catalysis lead to the hydrolysis of alkoxysilane functions and the production of silanol (Si-OH) groups. An acidic medium (e.g. pH 1-4) hinders the formation of oxo-bridges from the subsequent condensation of silanol groups, whereas a more basic medium (e.g. pH 5-10) produces rapid condensation of silanol groups resulting in gel formation. Even after the gelation point, the structure and properties of the gel continue to change during the drying process. One reason for these changes is that polycondensation reactions are still taking place in the solid amorphous phase, and as a result, cross-linking continues. Spontaneous shrinkage of the gel and the resulting expulsion of pore liquid also occur. This expulsion is caused primarily by the formation of new bonds via polycondensation and the resultant contraction of the gel network and is called syneresis. The strength of the gel increases and pores become smaller as these aging processes take place. Nevertheless, some of the water and methanol generated as a result of hydrolysis is retained in the gel during this stage; an aged gel is thus a solid-state glassy material that contains a trapped aqueous phase. The polycondensation process continues during aging, and a porous matrix is formed around the protein molecule, trapping it inside. Such physical entrapment of the protein is functionally non-invasive and preserves the integrity and directional homogeneity of the protein surface microstructure. Although the mode of entrapment of biomolecules in sol-gels is primarily physical in that the pore size of the sol-gels is equal to or smaller than the protein, there are other interactions that also contribute to the interaction of the protein with the sol-gel matrix. These include ionic interaction between charged groups of proteins with ionizable groups in the matrix, hydrogen bonding, non-polar interactions (Van Der Waals forces) and even covalent bonds. The entrapped protein is stabilized not only by a physical barrier, but also by immobilizing the protein in a silica gel matrix. Protein stabilization in the silica gel matrix is a consequence of prevention or minimization of the interaction between protein segments due to immobilization, the collision between protein segment and the hydrated silica surface on which the protein is immobilized, and the electrostatic interaction between proteins and other counter-charged macromolecules. The present invention is directed to a method for preparing enzyme-containing silica sol-gels, wherein one or more enzymes are stably entrapped within the gels and are released from the gels within less than an hour when the gels are placed in a solution having water percentage by weight of 86% or more. The method comprises the steps of (a) mixing (i) one or more aminoalkylsilane precursors selecting from the group consisting of bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane, (ii) one or more alkoxysilane precursors selected from the group consisting of dialkyldialkoxysilane and alkyltrialkoxysilane, and (iii) a first aqueous solution to hydrolyze the silane precursors to silicate or organosiloxane sols; (b) adding to the sols a second aqueous solution containing one or more enzymes; and (c) incubating (b) to form gels. Optionally, step (a) further comprises mixing (iv) one or more negatively charged organosilane precursors together with (i), (ii), and (iii). Step (a) in general is carried in an acidic pH (e.g., pH 1-6) or a basic pH (e.g., pH 8-13) to facilitate the formation of sols. A preferred acidic pH is for example is pH 1-5, or pH 1.5-4. A preferred basic pH is for example is pH 9-12. Alternatively, the method comprises the steps of (a) mixing one or more aminoalkylsilane precursors selecting from the group consisting of bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane, with a first aqueous solution to form a first sol; (b) mixing one or more alkoxysilane precursors selected from the group consisting of dialkyldialkoxysilane and alkyltrialkoxysilane with a second aqueous solution to form a second sol; (c) mixing the first sol, the second sol, and a third aqueous solution containing one or more enzymes, and (d) incubating (c) to form gels. In step (a) or (b), the aminoalkylsilane precursors or the alkoxysilane precursors can be hydrolyzed to form a silicate or organosiloxane sol at either an acidic pH (pH 1-6) or a basic pH (pH 8-13). A preferred acidic pH is, for example, pH 1-5, or pH 1.5-4. A preferred basic pH is, for example, pH 9-12. An aqueous solution that contains one or more enzymes is usually buffered at a pH that stabilizes the enzymes or does not denature the enzymes. For example, an aqueous enzyme solution buffered at pH 4-10, preferably pH 5-9, is suitable for use in this invention. A silicate sol is defined as a stable colloidal solution of silicate oligomers where the particle size is in the nanometer range. Silicate sols can undergo gelation or precipitation when exposed to a change in pH or a catalyst (Her, R.K. 'The Chemistry of Silica' (Wiley, 1979); Brinker, C.J. and Scherer, G.W. 'Sol Gel Science: The Physics and Chemistry ofSol- Gel Processing' (Academic press, 1990)). In the present method for preparing sol-gels, the order for mixing sols or enzymes to form gels is not important. However, it is important that the enzymes are not exposed to an extreme pH such as outside of pH 4-10 or 5-9 during the process. The formation of sols can be accelerated by sonicating the precursors in the acidic solution, for example, for 30 minutes to an hour. The time to form gels varies from a few minutes to many hours. The gelation time usually takes 20 minutes to 1 hour. After gels are formed, prolonged curing and drying of sol-gels often result in a reduction in both enzyme leakage during storage and enzyme release upon dilution into water. Sol-gels that are optimally cured and dried have low enzyme leakage during storage and high enzyme release upon dilution into water. After gelation, sol-gels of the present invention are often cured for about 1 day to 1 week before formulated into a detergent formulation. Sol-gels can be in various forms. In one embodiment of the invention, sol-gels are in the form of a fine powder, slurry, a microemulsion, or an emulsified suspension. In another embodiment of the invention, sol-gels can be suspended in liquids. In yet another embodiment of the invention, sol-gels can be incorporated into granules for addition to powdered product. Sol-gels can be stored as crushed powders or pastes or any other as form mentioned above. Controlled monodispersed sol-gel microparticles can be prepared based on emulsion techniques known to a skilled person, for example, see Osseo-Asare, K. et al. (1990) "Synthesis of nanosize particles in reverse microemulsions" Ceramic Trans., 12: 3-16; Osseo-Asare, K et al. (2000) "Silica. Hydrolysis of silicon alkoxides in microemulsions" Surfactant Science Series (2000), 92: 147-188; and Balducci, L. Ungarelli, R. (1995) "Process for manufacturing porous spherical silica xerogels for catalyst carrier" EP 653378 Bl. Sol-gel microparticles can also be prepared by mechanically crushing a sol-gel monolith, which usually results in highly polydispersed distributions in particle sizes. The starting materials for enzyme-containing silica gels that are responsive to water percentage change include a combination of one or more aminoalkylsilane precursors and one or more alkoxysilane precursors. Pure alkoxysilane sol-gels derived from silicate precursors such as tetramethylorthosilicate (TMOS), and tetraethylorthosilicate (TEOS), do not release the entrapped protein in response to a triggering event of increasing the water content. Adding an aminoalkylsilane precursor makes the gel responsive or sensitive to water percentage changes. In an alternative embodiment, the alkoxysilane precursors can be fully or partially replaced by metal salts of organosilanes. In this embodiment, the Si-OH groups capable of condensation with gel formation are generated by the protonation of Si-O-metal groups, such as an alkylsiliconate, e.g. sodium methylsiliconate, MeSi(ONa)₃. In a further embodiment, the starting materials include a combination of alkoxysilane precursors, metal salts of organosilanes, and aminoalkylsilane precursors. The aminoalkylsilane precursors useful as the starting material for the present invention include bis[(trimethoxysilyl)propyl] ethylenediamine (EnTMOS), bis[3-trimethoxysilyl)propyl]amine, (ATMOS), and bis(methyldiethoxysilylpropyl)amine, and 3-aminopropyltrimethoxysilane (3-APTS). The alkoxysilane precursors useful as the starting material for the present invention include dimethyldimethoxysilane (DMDS), methyltrimethoxysilane (MTMOS). In one embodiment of the invention, the starting material optionally comprises a negatively charged silane precursor such as trialkoxysilylalkylsuccinic anhydride, trihydroxysilylalkylphosphonate, trihydroxysilylalkylsulfonic acid or carboxyalkylsilanetriol. The negative charge added in the silica gel matrix modulates the interaction of the matrix with the environmental medium, and often increases the release rate of enzyme upon the dilution of the silica gel with water. The negatively charged organosilane precursors useful as a starting materials include 3-(triethoxysiiyl)propylsuccinic anhydride, 3- trihydroxysilylpropylmethylphosphonate, and 3-(trihydroxysilyl)-l-propanesulfonic acid, or carboxyethylsilanetriol. In another embodiment of the invention, one or more additives such as disaccharides, polysaccharides, water soluble or dispersible polymers, i.e., polyvinyl alcohols, polyethylene glycol, or polypropylene glycol, can be added during the preparation of sol-gels. The additives can be added in any step prior to the gelation. Polysaccharides, for example, include starch, pectin, sodium, alginate, or carrageenan. A useful additive is sucrose. Sucrose helps formation of the gel network by hydrogen bonding. Sucrose also adds hydrophihcity to the gel system. The moisture retention helps in releasing the encapsulated enzyme, upon the dilution of the gel system in water. Swelling and shrinking capacity of the silica gel matrices is an important criterion on which this invention is based. One of the several mechanisms for enzyme release from sol- gels responsive to environmental water percentage is that upon dilution in water, swelling of the gel results in expansion of the micro pores in which the enzyme molecules are entrapped. This expansion of the micro pores allows for the diffusion and subsequent release of the enzyme from the sol-gel into the surrounding medium. Swelling is the volume expansion of a monolilthic gel through absorption of water (like a dry sponge absorbing water). Swelling and shrinking can be determined by weighing the gel before and after its exposure to water. Swelling can be reported as an increase in mass and shrinking can be reported as a decrease in mass. Alternatively, swelling and shrinking can also be determined by measuring the volume of the gel before and after its exposure to water. Measurement by weight is easier than measurement by volume. Swelling/shrinking of sol-gels is one of several mechanisms that allow for the release of enzyme from sol-gels upon a change in the surrounding fluid's water activity. Other mechanisms for controlling protein's release from sol-gels include hydrophobic interactions, hydrophilic interactions (i.e. hydrogen bonding) and ionic interactions, between the protein and sol-gels. Further mechanical mechanisms that control protein's release include agitation and shear. Sol-gels obtained from the starting materials of the present invention exhibit bulk volume changes and generate active mechanical responses when the surrounding environment changes. The enlarged pores obtained due to aminoalkoxysilane precursors, such as bis[3-(trimethoxysilyl)propyl]ethylenediamine (enTMOS), or (CH₃O)₃Si(CH₂)₃NH(CH₂)₃Si(OCH₃)₃ (ATMOS), with long chain spacer units, help in retaining aqueous phase in the porous network, which in turn makes the swelling and shrinking mechanism more pronounced. The hydrophilic side chain of the precursor endows pH sensitivity to the gel system. It is observed that the bulk gel undergoes swelling in acidic pH and shrinking in basic pH. The swelling of the gels is attributed to protonation of the side chain amino groups in low pH; the gel absorbs water molecules to minimize the repulsion between the chains. Enzymes suitable for entrapment in the sol-gel system can be any enzymes. Enzymes include but are not limited to commercially available types, improved types, recombinant types, wild types, variants not found in nature, and mixtures thereof. Preferred enzymes are detergent enzymes used in laundry detergents, fabric care products, or dishwasher detergents. Suitable enzymes include hydrolases, cutinases, oxidases, transferases, reductases, hemicellulases, esterases, isomerases, pectinases, lactases, peroxidases, laccases, pectinases, catalases, and mixtures thereof. Hydrolases hydrolyze substrates, e.g., stains, and are used in laundry detergents, dish detergents, and fabric care products. Hydrolases include, but are not limited to, proteases (bacterial, fungal, acid, neutral or alkaline), amylases (alpha or beta), lipases, mannanases, cellulases, and mixtures thereof. Suitable enzymes for this invention also include those sold by Genencor International under the trade names Purafect, Purastar, Properase, Puradax, Clarase, Multifect, Maxacal, Maxapem, and Maxamyl (U.S. Patent No. 4,760,025 and WO 91/06637); and those sold by Novo Industries A/S (Denmark) under the trade names Alcalase, Savinase, Primase, Durazyme, Duramyl, Lipolase, and Termamyl. Suitable proteases are subtilisins, produced by Bacillus species. Another suitable enzyme is cellulase and particularly cellulase or cellulase components isolated from Trichoderma reesei, such as found in the product Clazinase. Amylases such as alpha amylases obtained from Bacillus licheniformis are also suitable enzymes. Proteases are especially suitable for entrapment in a sol- gel system because of their hydrolytic action upon other enzymes and also their autolytic or self-proteolytic action. Proteases and other enzymes are typically produced by aerobic fermentation of bacteria or fungi. These enzymes are generally secreted as extracellular proteins, but in some cases, enzymes can be isolated from the cell membrane or from within the cell by chemical, enzymatic or physical disruption. Commercially, the cells and cell debris are removed by processes such as centrifugation or filtration through porous media, often with the aid of flocculation agents. Prior to the sol-gel process, enzymes are preferably concentrated by removing water and low molecular weight species such as salts or peptides, e.g., by ultrafiltration, evaporation, precipitation or extraction. Generally, ultrafiltration or tangential flow filtration through polymeric or ceramic membranes is a preferred practical or economical route. The present invention provides an enzyme-containing sol-gel composition that is responsive to water percentage change. The present invention provides enzyme-containing sol-gels prepared from starting materials comprising one or more aminoalkylsilane precursors selecting from the group consisting of bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane, and one or more alkoxysilane precursors selected from the group consisting of dialkyldialkoxysilane and alkyltrialkoxysilane, wherein one or more enzymes are contained within the sol-gels, and at least 30% of said one or more enzymes are released from the sol-gels within an hour upon diluting the sol-gels into a solution that increases the water percentage by weight to 86% or higher. Water percentage as used herein is the percentage of water by weight in a solution. For example, a solution having a water percentage of 100% is pure water; a solution having a water percentage of 60% is a solution having 60% water by weight. One or more enzymes can be entrapped in the sol-gel system. The entrapped enzymes in general have an improved stability over the free, non-entrapped enzymes. The enzymes in the sol-gel composition are stable during storage in that they maintain at least 60%, 70%, or 80%, preferably at least 85%>, and more preferably at least 90%, of activities for 7 days at room temperature. The enzymes are thermally stable during storage in that they maintain at least 60%, 70%, or 80%, preferably at least 85%, and more preferably at least 90%, of activities for 2 days at elevated temperature such as 37 °C. The enzymes also stay within the sol-gels with less than 20%, preferably less than 15%, preferably less than 10%, and more preferably less than 5% leakage out of sol-gels for 7 days. Upon dilution of the sol-gel into a solution having a water percentage greater than 86%, the enzymes are quickly released into the solution. For example, at least 30% of enzymes are released from the sol-gel into the solution within one hour. The present invention provides a liquid detergent, a liquid soap or a shampoo formulation comprising the enzyme-containing sol-gels. The formulation is prepared by mixing the enzyme-containing sol-gels with the liquid detergent, the liquid soap or the shampoo. For example, sol-gels are crushed and a detergent is added to the crushed powders to form a viscous detergent/sol-gel paste. The enzymes are stable during storage in the formulation and are quickly released from the gels upon diluting the formulation in water. The enzymes are stable during storage in the formulation in that they maintain at least 60%, 70%), or 80%), preferably at least 85%, and more preferably at least 90%, of activities for 7 days at room temperature. The enzymes are thermally stable during storage in the formulation in that they maintain at least 60%, 70%, or 80%, preferably at least 85%, and more preferably at least 90%, of activities for 2 days at elevated temperature such as 37 °C. The enzymes also stay within the sol-gels with less than 20%, preferably less than 15%, preferably less than 10%, and more preferably less than 5% leakage out of sol-gels into the liquid detergent, liquid soap or shampoo formulation for 7 days. The liquid detergent, liquid soap or shampoo formulation in general has a water percentage (by weight) of 70% or lower, or 40%> or lower, or 30% or lower. For example, a "concentrated" or "compact" liquid detergent typically has 30-45 % water and a "dilute" liquid detergent typically has greater than 50-60% water, sometimes 70% water by weight. A common liquid detergent, TIDE^® (Procter & Gamble, Cincinnati, OH), can be classified as a compact liquid detergent. The enzymes in the formulation are released when the formulation is diluted in water to a water percentage by weight of 86% or higher, preferably 90% or higher, more preferably 95%) or higher, more preferably 98% or higher, and most preferably 99% or higher. During use such as in a laundry washing cycle, the formulation is diluted at least 5 fold, often 10, 100, or even 1000 fold in water. Table 1 shows the water percentage of a formulation before and after dilution in water. The present formulation comprises a stable enzyme-entrapped gel system. Upon dilution of the formulation into water, at least 30% (preferably 40%, 50%, 60%, 70%, or 80%) or the enzyme is released from the gel system into water within 1 hour, preferably 30 minutes, more preferably 10 minutes, more preferably 5 minutes, more preferably 2 minutes, and most preferably 1 minute.

Table 1. Water Percentage (by Weight) Before and After Dilution. Initial Water % Final Water % After Dilution

The present invention further provides a powder detergent formulation comprising the enzyme-containing sol-gels. In general, enzymes are more stable in a powder detergent form than in a liquid detergent form. However, a bleach-containing detergent powder is harmful to the enzymes. In addition, some detergent powders are hygroscopic and have a tendency to absorb water during storage in humid climates; which creates stability problems for the enzymes. A powder detergent formulation comprising the enzyme-containing sol-gels improves the enzyme stability. Sol-gels can be granulated by a variety of enzyme granulation processes, e.g., using fluid bed technology to spray-coat a slurry of the sol-gel as a layer on a core; mixing the sol-gel entrapped enzyme into a paste prior to drum granulation, wet granulation or extrusion, etc. The sol-gel granules are then mixed with the detergent powder to form a powder detergent formulation comprising the enzyme-containing sol-gels. The enzymes are stable during storage in such a formulation and are released from the gels upon diluting the formulation in water. In the present invention, silica gel matrices are used as vehicles for controlling the release of proteins such as enzymes used in cleaning products such as liquid detergents, soaps, and shampoos; the protein release is triggered by a dilution event that increases the water content. The gel matrices not only improve the thermal stability of enzymes but also prevent the enzyme denaturation due to proteolysis/autolysis or interference from other components of the cleaning products. Multi-enzyme detergent formulations are prepared by including different enzyme- entrapped silica gel matrixes in the detergent formulations, in which different enzymes are separated from each other by the gel matrixes, thus the proteolysis of the enzymes are prevented. By using a suitable gel matrix, a control over the release of the enzyme into the detergent formulation during the wash cycle can be achieved. For example, by choosing a suitable gel matrix, cellulases that are used as fabric softeners can be released during the last few minutes of the wash cycle.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting. EXAMPLES

Example 1. Preparation of SP-enTMOS gels

Preparation of SP sol: To 0.3 mL of CH₃OH, 0.18 mL of H₂O , 0.06 mL of HC1 (0.04 M) and 0.45 mL of 3-(triethoxysilyl)propylsuccinic anhydride (SP) were added. The mixture was sonicated for an hour to obtain the SP sol.

Preparation of DMDS sol: To 3 mL of CH₃OH, 0.8 mL of H₂O, 0.4 mL of HC1 (0.04 M), followed by 1 mL of dimethyldimethoxysilane were added and sonicated for 45 minutes. While continuously stirring, to 0.3 mL of DMDS sol, 0.44 mL of SP sol, 0.69 mL of bis- [3-(trimethoxysilyl)-propyl]ethylenediamine and 0.5 mL of stock protease (30mg/mL subtilisin, a Y217L protease variant derived from Bacillus amyloliquefaciens, as described in U.S. Patent No. 5,700,676, in 35-45% propylene glycol and 8-12% sodium acetate) were added. The sol/precursors were added in the order in which they were mentioned above. The gelation period was observed to be within 30 minutes. The formed gel was aged for about 24 hours prior to carrying out the release experiments in the detergent.

Example 2. Preparation of SP-ATMOS gels SP sol and DMDS sol were prepared as Example 1. Three different SP-ATMOS gels with varying amounts of SP sol and bis(trimethoxy silylpropyl)amine were prepared. The sol/precursors were added while continuously stirring in the order - CH₃OH, SP sol, bis(trimethoxysilylpropyl)amine, DMDS sol and the protease. The volumes of the various components in the gels is given below:

SP-ATMOS (0.3:1) - 0.5 mL CH₃OH, 0.2 mL of SP sol, 0.345 mL of bis(trimethoxysilylpropyl) amine, 0.5 mL of DMDS sol and 0.5 mL of stock protease (30mg/mL subtilisin, a Y217L protease variant derived from Bacillus amyloliquefaciens, as described in U.S. Patent No. 5,700,676, in 35-45% propylene glycol and 8-12% sodium acetate).

SP-ATMOS (0.3:0.6) - 0.5 mL CH₃OH, 0.2 mL of SP sol, 0.207 mL of bis(trimethoxysilylpropyl)amine, 0.5 mL of DMDS sol and 0.5 mL of stock protease. SP-ATMOS (0.35:0.6) - 0.5 mL CH₃OH, 0.233 mL of SP sol, 0.207 mL of bis(trimethoxysilylpropyl)amine, 0.5 mL of DMDS sol and 0.5 mL of stock protease. The gelation period was observed to be within 30 minutes. The formed gels were aged for about 24 hours prior to carrying out the release experiments in the detergent.

Example 3. Preparation of SP-AMPTMOS gels SP sol was prepared as Example 1.

Preparation of DMDS* sol To 8 mL of CH₃OH, 6 mL of H₂O, 0.8 mL of HC1 (0.04 M), followed by 2 mL of dimethyldimethoxysilane were added and sonicated for 45 minutes.

Preparation of MTMOS sol To 6 mL of H₂O, 0.1 mL of HC1 (0.04 M) and 9 mL of methyltrimethoxysilane were added and the mixture was sonicated for 45 minutes to obtain MTMOS sol. While continuously stirring 0.2 mL SP sol, 0.45 mL DMDS sol, 0.45 mL MTMOS sol, 0.345 mL 3-aminopropyltrimethoxysilane and 0.5 mL protease were added in the order in which they are mentioned. The gelation period was observed to be around 30 minutes. Gels were aged for 24 hours before carrying out the release experiments. Example 4. Preparation of Su 1/0.45 and Su 1/0.6 gels

Abbreviations used:

AMPTMOS = (3-aminopropyl)trimethoxysilane DMDS = dimethyldimethoxysilane MTMOS = methyltrimethoxysilane

DMDS sol:

2 ml DMDS + 8 ml methanol + 6 ml water + 0.8 ml HC1 (0.04M), sonicate for 50 minutes MTMOS sol:

9 ml MTMOS + 6 ml water + 0.1 ml HC1 (0.04M), sonicate for 50 minutes

Compositions Su 1/ 0.45 : (0.45 ml) DMDS sol + (0.45 ml ) MTMOS sol + 0.01 g sucrose + (0.345 ml) AMPTMOS

+ 0.5 ml stock protease (30mg/mL subtilisin, a Y217L protease variant derived from Bacillus amyloliquefaciens, as described in U.S. Patent No. 5,700,676, in 35-45% propylene glycol and 8-12%) sodium acetate).

Su 1/ 0.6 :

(0.6 ml) DMDS sol + (0.6 ml ) MTMOS sol + 0.01 g sucrose + (0.345 ml) AMPTMOS + 0.5 ml stock protease

Method of preparation DMDS and MTMOS sols were prepared according to Example 3. To 0.45ml / 0.6 ml

(0.45 in Sul/0.45 and 0.6 in Su 1/0.6) of DMDS sol, 0.45ml / 0.6 ml (0.45 in Sul/0.45 and 0.6 in Su 1/0.6) of MTMOS sol was added. In the mixture, 0.01 g of sucrose was dissolved. This was followed by the addition of AMPTMOS and finally protease. All the components were added while continuously stirring. Gels formed in 30-45 minutes. Gels were aged for about a day before adding to detergent.

Example 5. Thermal stability of enzyme A set of negatively charged SP-ATMOS (-ve) gels each containing 0.25 ml of protease (8.5 mg) were prepared according to Example 2, SP-ATMOS (0.35:0.6). The gel monolith was prepared in the bulb of a polyethylene transfer pipette. The obtained monolith was cylindrical in shape. After aging the gel for a day, the gels were immersed in 3 ml of a commercially available detergent TIDE^®, which had been heated at 90°C for one hour to deactivate the enzymes contained therein. One sample was left in oven at 39°C and another was left at room temperature (RT). A control experiment was also carried out by incubating 0.25 ml of the same free protease (which had been formulated for improved stability) at 39°C and RT. The protease enzyme activity was measured by carrying out the assay with subtrate N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA) at 25°C on a Shimadzu RF-5300 (PC) Series Spectrofluorophotometer. The enzyme solution was first diluted in 10 mM MES buffer (pH 5.5) containing 10 mM CaCl₂ and 0.005% Tween 80. The assay was carried out in lmL of 100 mM Tris buffer (pH 8.6) containing 0.005%) Tween 80 and 10 μL substrate solution (160 mM suc-AAPF-pNA in DMSO). The rate of hydrolysis of the substrate, which indicates the enzyme activity, was monitored by measuring the rate of change of absorbance at wavelength 410 nm for the initial 0-15 seconds. The enzyme activity in the SP-ATMOS (-ve) gel was measured after diluting the enzyme-containing detergent 1 : 10 in water for three hours. The results are shown in Table 2.

Table 2.

SPA (-ve) gel showed a 43.79%) active enzyme, which is an improvement over the free enzyme (31.86%) upon treatment at 39°C for 21 hours.

Example 6. Swelling and Shrinking Properties of SP-enTOMS and SP-ATMOS Gels. SP-enTMOS gels were prepared according to Example 1. The various compositions mentioned in the figure differ in the volume of DMDS sol used (0.3, 0.5, 0.7 and 0.9 ml). SP-ATMOS gels were prepared according to Example 2 for the gel system SP- ATMOS ( 0.3:0.6 ). The various compositions mentioned differ in the volume of DMDS sol used ( 0.5, 0.7 and 0.9 ml). The gels were aged for a day then soaked in water or in detergent (TIDE^®), which had been heated at 90°C for one hour to deactivate the enzymes contained therein. The gels were periodically weighed. The difference in weight with respect to time in both media is attributed to swelling/ shrinking in the two media. The SP-enTMOS and SP-ATMOS gels show features of swelling in water and shrinking in detergent, which are useful for the current application. The % weight change patterns for the SP-enTMOS and SP-ATMOS are given in Figures 1 and 2.

Example 7. Enzyme Release Experiment from Su 1/0.45 Gel. Su 1/0.45 gel was prepared according to Example 4 except that it is a four-time scaled-up version with the compositions: (1.8ml) DMDS sol + (1.8 ml) MTMOS sol + 0.04 g sucrose + (1.38 ml) AMPTMOS + 2 ml stock protease (30mg/mL subtilisin, a Y217L protease variant derived from Bacillus amyloliquefaciens, as described in U.S. Patent No. 5,700,676, in 35-45%) propylene glycol and 8-12% sodium acetate). The gel containing 70 mg of enzyme (2 ml) was first aged for a day, then soaked for one day in 10 mL of detergent TIDE , which had been heated at 90°C for one hour to deactivate the enzymes contained therein. The enzyme that leaked out into the surrounding detergent medium was monitored by the assay similar to that described in Example 5. 10 μL of the supernatant detergent and 10 μL of the substrate (suc-AAPF-pNA) were added to 1 ml of lOOmM Tris buffer (pH 8.5) maintained at 25°C. The rate of hydrolysis was measured on a spectrophotometer at 410 nm for initial 20 seconds. The leakage of the enzyme into the detergent after 24 hours was observed to be about 1.2%. After a day, the system containing the gel and the detergent was diluted with 500 ml of water and the enzyme released was measured. The gel system released about 78% of the total enzyme after its dilution in water.

Example 8. Enzyme release experiment from Su 1/0.6 Gel. Su 1/0.6 gel was prepared according to Example 4 except it is a 4-time scaled-up version. The compositions were (2.4 ml) DMDS sol + (2.4 ml) MTMOS sol + 0.04 g sucrose + (1.38 ml) AMPTMOS + 2 ml stock protease (30mg/mL subtilisin, a Y217L protease variant derived from Bacillus amyloliquefaciens, as described in U.S. Patent No. 5,700,676, in 35-45%) propylene glycol and 8-12% sodium acetate). The gel containing 70 mg of enzyme (2 mL) was aged for a day and soaked for 5 days in 10 mL of detergent TIDE^®, which had been heated at 90°C for one hour to deactivate the enzymes contained therein. The enzyme that leaked out into the surrounding detergent medium was monitored as described in Example 7. The leakage of the enzyme into the detergent after 24 hours was observed to be about 10% of the total protease initially present within the gel. After 5 days, the gel and detergent were subjected to a dilution trigger (50 times dilution with respect to detergent) into water under mechanical agitation and the enzyme released was measured. Ideally at 100%> release, the system should give an enzyme assay value of 70 mg. After being in detergent for 5 days, the Su 1/0.6 gel system released about 60% enzyme upon dilution trigger into water.

Example 9. Preparation of dye-containing Su 1/0.45 gels Silica sol-gel containing a dye was prepared. This is a visual model that can easily demonstrate the release of the active component from the gel.

Abbreviations used: AMPTMOS = (3-aminopropyl)trimethoxysilane DMDS = dimethyldimethoxysilane MTMOS = methyltrimethoxysilane

DMDS sol: 2 ml DMDS + 8 ml methanol + 6 ml water + 0.8 ml HCl (0.04M), sonicate for 50 minutes.

MTMOS sol:

9 ml MTMOS + 6 ml water + 0.1 ml HCl (0.04M), sonicate for 50 minutes

Compositions Su 1/ 0.45 :

(0.45 ml) DMDS sol + (0.45 ml ) MTMOS sol + 0.01 g sucrose + (0.345 ml) AMPTMOS + 0.75 mL of Chicago Sky Blue dye (0.02 mg/mL in water) Method of preparation DMDS and MTMOS sols were prepared according to Example 3. To 0.45 ml of DMDS sol, 0.45ml of MTMOS sol was added. In the mixture, 0.01 g of sucrose was dissolved. This was followed by the addition of AMPTMOS and finally the dye solution. All the components were added while continuously stirring. Gels formed in 30-45 minutes. The obtained gels were blue translucent gels with a firm appearance. When about 100 mL of water was added to about 2 mL of the dye-containing gels with stirring, it was observed visually that the dye was released into water. Another experiment was performed by first storing the gels in detergent (TIDE^®) for 2 hours. Most of the detergent was poured off from the gels, and then water was added to the gels with stirring. The dilution factor in water was over 10-fold. The release of dye from gels into water was visually observed.

Example 10. Preparation of sol-gels with PEG additive. SP sol and DMDS sol were prepared according to Example 1. While continuously stirring, to 0.15 mL of DMDS sol (0.225 x 10^-3 moles), 0.22 mL of SP sol (0.335 x 10^~3 moles), and 0.345 mL (0.545 x 10^~3 moles) of bis-[3- (trimethoxysilyl)-propyl]ethylenediamine, 0.05 mL of polyethylene glycol (PEG) was added, followed by 0.25 mL of stock protease (30mg/mL subtilisin, a Y217L protease variant derived from Bacillus amyloliquefaciens, in 35-45% propylene glycol and 8-12% sodium acetate). The gel monolith was prepared in the bulb of a polyethylene transfer pipette. The gelation period was between 15- 30 minutes. The obtained monolith was cylindrical in shape.

Example 11. Enzyme leakage during storage in detergent and enzyme release upon dilution in water. Sp-enTMOS gel was prepared according to Example 1. ESP gel was prepared similarly to Sp-enTMOS gel, except 0.05 ml of polyethylene glycol was added to the sol composition of Sp-enTMOS gel before the addition of enzyme. Negatively charged SP-ATMOS (-ve) gel containing 0.25 ml of protease (8.5 mg) was prepared according to Example 2, SP-ATMOS (0.35:0.6). ESF-NS gel is a non-sonicated composition. To 0.125 ml of enTMOS precursor, 0.1ml of SP precursor was added. The precursors initially formed two phases but eventually homogenized. To the homogenous mixture, 1 ml of formate solution was added followed by 0.25 ml of enzyme. Each of Sp-enTMOS, ESP, ESF-NS and SP-ATMOS (-ve) gel monolith was immersed in TIDE^®, which had been heated at 90°C for one hour to deactivate the enzymes contained therein. The gels were stored for 1-4 weeks in detergent without rotation during the storage period. At the end of each storage period, the samples were rotated at 8 m for 1 minute to mix any enzyme that had leaked out from the gels into the detergent, and then the detergent was tested for the enzyme activity. The results of percent enzyme leaked from gels (Sp-enTMOS, ESP, ESF-NS and SP-ATMOS (-ve)) into detergent during static storage in detergent for 1, 2, 3, or 4 weeks are shown in Figure 3. Each gel-containing detergent formulation was diluted 10-fold in water and agitated at 8 φm for about an hour. After storage in the detergent for 1 , 2, 3, or 4 weeks, each of Sp- enTMOS, ESP, ESF-NS and SP-ATMOS (-ve) gel was also tested for their responsiveness to dilution in water. Each gel-containing detergent formulation was diluted 10-fold in water and agitated at 8 φm for about an hour. The results of percent enzyme released from the gels upon dilution of the gel-containing detergent in water within one hour are shown in Figure 4.

Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the scope of the invention.

Claims

What is Claimed is:

1. Enzyme-containing sol-gels prepared from starting materials comprising one or more aminoalkylsilane precursors selecting from the group consisting of bis(trialkoxyalkylsilane) amine, bis(triaιkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane, and one or more alkoxysilane precursors selected from the group consisting of dialkyldialkoxysilane and alkyltrialkoxysilane, wherein one or more enzymes are contained within the sol-gels, and at least 30% of said one or more enzymes are released from the sol-gels within an hour upon diluting the sol-gels into a solution that increases the water percentage by weight to 86%> or higher.

2. The enzyme-containing sol-gels according to Claim 1, wherein said bis(trialkoxyalkylsilane)alkylenediamine is bis[(trimethoxysilyl)propyl] ethyl enediamine.

3. The enzyme-containing sol-gels according to Claim 1, wherein said bis(trialkoxyalkylsilane)amine is bis[3-trimethoxysilyl)-propyl]amine.

4. The enzyme-containing sol-gels according to Claim 1, wherein said bis(dialkyldialkoxysilane)amine is bis(methyldiethoxysilylpropyl)amine.

5. The enzyme-containing sol-gels according to Claim 1, wherein said dialkyldialkoxysilane is dimethyldimethoxysilane.

6. The enzyme-containing sol-gels according to Claim 1, wherein said alkyltrialkoxysilane is methyl(trimethoxysilane).

7. The enzyme-containing sol-gels according to Claim 1, wherein said aminoalkyltrialkoxysilane is 3-aminopropyltrimethoxysilane.

8. The enzyme-containing sol-gels according to Claim 1, wherein the starting materials further comprise a negatively charged silane precursor.

9. The enzyme-containing sol-gels according to Claim 8, wherein the negatively charged silica precursor is trialkoxysilylalkyl succinic anhydride, trihydroxysilylalkylphosphonate, trihydroxysilylalkylsulfonic acid, or carboxyalkoxysilanetriol.

10. The enzyme-containing sol-gels according to Claim 9, wherein the negatively charged silica precursor is 3-(triethoxysilyl)propylsuccinic anhydride, 3-trihydroxysilylpropylmethyl phosphonate, 3-(trihydroxysilyl)-l-propanesulfonic acid, or carboxyethoxysilanetriol.

11. The enzyme-containing sol-gels according to Claim 1 , wherein the starting materials further comprises one or more disaccharides, polysaccharides, polyvinyl alcohols, polyethylene glycol, or polypropylene glycol.

12. The Enzyme-containing sol-gels according to Claim 1, wherein said one or more enzymes are stable for at least a week at temperature between 22-28 °C.

13. A liquid detergent formulation comprising enzyme-containing sol-gels prepared from starting materials comprising one or more aminoalkylsilane precursors selecting from the group consisting of bis(trialkoxyalkylsilane)amine, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, and aminoalkyltrialkoxysilane, and one or more alkoxysilane precursors selected from the group consisting of dialkyldialkoxysilane and alkyltrialkoxysilane, wherein one or more enzymes are contained within the sol-gels.

14. The liquid detergent formulation according to Claim 13, wherein said bis(trialkoxyalkylsilane)alkylenediamine is bis[(trimethoxysilyl)propyl]ethylenediamine.

15. The liquid detergent formulation according to Claim 13, wherein said bis(trialkoxyalkylsilane)amine is bis[3-trimethoxysilyl)-propyl]amine.

16. The liquid detergent formulation according to Claim 13, wherein said bis(dialkyldialkoxysilane)amine is bis(methyldiethoxysilylpropyl)amine.

17. The liquid detergent formulation according to Claim 13, wherein said dialkyldialkoxysilane is dimethyldimethoxysilane.

18. The liquid detergent formulation according to Claim 13, wherein said alkyltrialkoxysilane is methyltrimethoxysilane).

19. The liquid detergent formulation according to Claim 13, wherein said aminoalkyltrialkoxysilane is 3-aminopropyltrimethoxysilane.

20. The liquid detergent formulation according to Claim 13, wherein the starting materials further comprise a negatively charged silane precursor.

21. The liquid detergent formulation according to Claim 20, wherein the negatively charged silica precursor is trialkoxysilylalkyl succinic anhydride, trihydroxysilylalkylphosphonate, trihydroxysilylalkylsulfonic acid or carboxyalkoxysilanetriol.

22. The liquid detergent formulation according to Claim 21 , wherein the negatively charged silica precursor is 3-(triethoxysilyl)propylsuccinic anhydride, 3- trihydroxysilylpropylmethyl phosphonate, 3-(trihydroxysilyl)-l-propanesulfonic acid, or carboxyethoxysilanetriol.

23. The liquid detergent formulation according to Claim 13, wherein the starting materials further comprises one or more disaccharides, polysaccharides, polyvinyl alcohols, polyethylene glycol, or polypropylene glycol.

24. The liquid detergent formulation according to Claim 13, wherein said one or more enzymes are stable for at least a week at temperature between 22-28 °C.

25. The liquid detergent formulation according to Claim 24, wherein less than 20%) of the one or more enzymes are released from the sol-gels when stored for one week at 22-28 °C.

26. The liquid detergent formulation according to Claim 24, wherein more than 30%) of the one or more enzymes are released from the sol-gels within one hour upon diluting the liquid detergent formulation into a solution that increases the water percentage by weight to 86%> or higher.

27. The liquid detergent formulation according to Claim 13 wherein said one or more enzymes are detergent enzymes used in laundry detergents, fabric care products, or dishwasher detergents.

28. The enzyme-containing gels according to Claim 27, wherein said detergent enzymes are selected from the group consisting of a protease, an amylase, a lipase, a cellulase, a mannanase, a ligninase, a pectinase, a peroxidase, and a catalase.

29. The enzyme-containing gels according to Claim 28, wherein said protease is a subtilisin.