WO2005028603A1

WO2005028603A1 - Silicate derived sol-gels sensitive to water content change

Info

Publication number: WO2005028603A1
Application number: PCT/US2004/030989
Authority: WO
Inventors: Nathaniel T. Becker; Dave C. Bakul; Kiranmayi Deshpande; Mark C. 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-entrapped 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 method for preparing such a composition comprises the steps of : (a) mixing a first aqueous solution with one or more alkali metal silicates or alkyl siliconate salts to reduce the pH to 12 or lower to form a first sol; (b) mixing a second aqueous solution with one or more organofunctional silane precursors to form a second sol; (c) mixing the first sol, the second sol, and a third aqueous solution containing one or more enzymes to form gels. 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

SILICATE DERIVED SOL-GELS SENSITIVE TO WATER CONTENT CHANGE

FIELD OF INVENTION The present invention relates to silicate 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 hydrolyse 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 autolysis 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, monellin, 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 tetramethylorthosilicate (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^Si- spacer-Si(OR ) , 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 is directed to a method for preparing enzyme-entrapped silica sol-gels, wherein one or more enzymes are stably entrapped within the gels and are released from the gels 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 a first aqueous solution with one or more alkali metal silicates or alkyl siliconate salts to reduce the pH to 12 or lower to form a first sol; (b) mixing a second aqueous solution with one or more organofunctional silane precursors selected from the group consisting of dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, or aminoalkylsilane to form a second sol; (c) mixing the first sol, the second sol, and an aqueous buffered solution containing one or more enzymes; and (d) incubating (c) to form gels. The present invention provides an enzyme-entrapped sol-gel composition that is sensitive to water percentage change. The present invention further provides a liquid detergent, a liquid soap or a shampoo formulation comprising an enzyme-containing sol-gel system. The enzymes are stable during storage in the formulation. The enzymes stay within the gels without significant leakage during the storage and are released from the gels upon diluting the formulation in water within less than an hour. 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. The present invention is based on use of silica 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/autolysis or interference from other components of the cleaning products. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the percentage of weight change at different time intervals after immersed in water or detergent. Figure 2 shows the amount of enzyme released from gels (a) when the gel is soaked in pH 7.85 buffer, and (b) after the gel is soaked in TIDE^® for one day, then subjected to a dilution trigger. Figure 3 shows the amounts of enzyme (mg/ml) released from gels having different additives (starch, pectin, alginate, and carageenan) after a dilution trigger at different time intervals. Figure 4 shows the percentage of enzyme released from gels that contain different additives (starch, pectin, alginate, and carageenan), at 10 minutes and 60 minutes after a dilution trigger. Figure 5 shows the amount of enzyme released from different gel systems. Figure 6 shows the percentage of enzyme released from silicate/siliconate (JA) and sodium silicate/starch (SS) gels upon a 10-fold dilution with water within an hour, after 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 silica 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 organofunctional silane precursors or neutralization of alkali metal silicates or alkyl siliconate salts. Further polymerization/chemical reaction/hydrolysis converts the sols into gels by chemically linking together the individual colloidal sol particles into monolithic gels. For example, a sol is first formed by hydrolysis of an alkoxysilane precursor followed by condensation to yield a polymeric oxo-bridged SiO₂ network. In the process, molecules of the corresponding alcohol are liberated. 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 stoichiometry 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 curing and 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 a first aqueous solution with one or more alkali metal silicates or alkyl siliconate salts to reduce the pH to 12 or lower to form a first sol; (b) mixing a second aqueous solution with one or more organofunctional silane precursors selected from the group consisting dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, or aminoalkylsilane so as to promote hydrolysis and 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. Optionally, the method further comprises the steps of hydrolyzing one or more negatively charged organosilane precursors to form a third sol, and mixing the third sol with the first sol, the second sol, and the aqueous solution containing one or more enzymes. In the above method, the first aqueous solution in step (a), which reduces the pH of the alkali metal silicates or alkyl siliconate salts to 12 or lower, can be an acid, e.g., phosphoric acid, citric acid, acetic acid, and hydrochloric acid, an acidic solution, or a low pH buffer. In step (b) or (c), the organofunctional silane precursors or the negatively charged organofunctional silane precursors can be hydrolyzed to form a second sol or a third 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. In another embodiment of the application, the method comprises the steps of: (a) mixing (i) a first aqueous solution, (ii) one or more alkali metal silicates or alkylsiliconate salts, and (iii) one or more organofunctional silane precursors such as dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, and aminoalkylsilane to form sols; (b) mixing the sols with 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. 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 of Sol- 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 a highly polydisperse distribution in particle size. Silicate precursors useful for the present invention include alkali metal silicates and alkyl siliconate salts. Alkali metal silicates include sodium silicates (e.g. sodium metasilicate, sodium orthosilicate and sodium silicate solutions), potassium silicates, and cesium silicates. Preferred alkali metal silicates are sodium silicates and potassium silicates.

The most preferred alkali metal silicates are sodium silicates. Sodium silicates are commercially available. For example, sodium metasilicate and sodium orthosilicate can be obtained from Gelest Inc. (Morrisville, PA). Sodium silicate solution (a solution of SiO and

NaOH) can be obtained from Sigma Aldrich. i Alkyl siliconate salts include sodium alkyl siliconate, potassium alkyl siliconate, and cesium alkyl siliconate. Preferred alkyl siliconate salts are sodium alkyl siliconate and potassium alkyl siliconate. The most preferred alkyl siliconate salt is sodium methyl siliconate. 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 alkyl siliconate, e.g. sodium methylsiliconate, MeSi(ONa) . Organofunctional silane precursors useful for the present invention include dialkyldialkoxysilanes, alkyltrialkoxysilanes, alkylsilanes, and aminoalkylsilanes. An example of an dialkyldialkoxysilane is dimethyldimethoxysilane (DMDS). An example of an alkyltrialkoxysilane is methyltrimethoxysilane (MTMOS). An example of an alkylsilane is aminopropylsilanetriol. Examples of aminoalkylsilanes are aminoalkyltrialkoxysilane, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, bis(trialkoxyalkylsilane)amine, and aminoalkylsilanetriol or its salts. An example of an aminoalkyltrialkoxysilane is 3-aminopropyltrirnethoxysilane (3-APTS). An example of a bis(trialkoxyalkylsilane)alkylenediamine is bis[(trimethoxysilyl)propyl]ethylenediamine

(EnTMOS). An example of a bis(dialkyldialkoxysilane)amine is bis(methyldiethoxysilylpropyl)amine. An example of a bis(trialkoxyalkylsilane)amine is bis[3-trimethoxysilyl)-propyl]amine (ATMOS). The negatively charged organosilane precursors useful for this invention, for example, include trialkoxysilylalkyl succinic anhydride, trihydroxysilylalkylphosphonate, trihydroxysilylalkyl sulfonic acid or carboxyalkylsilanetriol. Preferred negatively charged organosilane precursors are 3-(triethoxysilyl)propyl succinic anhydride, 3- trihydroxysilylpropylmethylphosphonate, 3-trialkoxysilylpropylmethylphosphonate, and 3-

(trihydroxysilyl)-l-propanesulfonic acid, carboxyalkylsilanetriol, or carboxyalkyltrialkoxysilane. In another embodiment of the invention, one or more additives such as disaccharides, polysaccharides, water soluble or dispersable 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 hydrophilicity to the gel system. The moisture retention helps in releasing the encapsulated enzyme, upon the dilution of the gel system in water. The starting materials for enzyme-containing silica gels that are responsive to water percentage change include a combination of one or more silicate precursors and one or more organofunctional silane 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 organofunctional silane precursor makes the gel responsive or sensitive to water percentage changes. 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 AS (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-entrapped sol-gel composition that is responsive to water percentage change. 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-entrapped sol-gel system. 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%o, 70%), or 80%o, 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%₀, 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 sodium silicate gels (Slaa) Preparation ofMTMOSsol. 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 a MTMOS sol. Silicate sol. To a solution of 1.2 ml H₃PO₄ (3M) and 0.5 ml H₂O, aqueous sodium silicate solution containing 0.75 ml sodium silicate solution (27%> w/w silica and 14%> w/w NaOH, Aldrich, WI) and 1.05 ml H₂O was added to form a silicate sol. To the silicate sol, 0.75 ml stock enzyme (30mg/mL subtilisin, a Y217 L protease variant derived from Bacillus amyloliquefaciens, as described in U.S. Patent No. 5,700,676, in 35-45%o propylene glycol and 8-12% sodium acetate) was added. This was followed by addition of 0.45 ml MTMOS sol. All the components were added dropwise while stirring the sols continuously. Gels were formed in about 45 minutes. The obtained gels were translucent and firm. A set of blank Slaa gels (without enzyme) was also prepared for carrying out the swelling and shrinking experiments.

Example 2. Swelling/Shrinking experiment The blank Slaa gels were subjected to the swelling and shrinking experiments. The gels were weighed at regular time intervals after keeping them immersed in water/detergent. The % weight change is calculated as the percentage change in the weight of the gel in the medium for that time interval as compared to the starting weight. A negative % weight change indicates shrinking of the gel matrix in that particular medium while a positive weight change indicates swelling. The results are shown in Figure 1, which indicates similar extent of % weight change when the gels were immersed in water and detergent. The results indicate that the gels shrank in both the water and detergent media.

Example 3. Release of enzyme from the Slaa gel upon a dilution trigger in water The enzyme-containing S 1 aa gel was soaked for a day in TIDE^® liquid detergent, which had been heated at 90°C for one hour to deactivate the enzymes contained therein. The following day, the gel-containing detergent was diluted in water, under mechanical agitation over a period of two hours. The amount of the enzyme released from gel at each time interval was determined by assaying the enzyme activity in the medium in which the enzyme release was carried out. The enzyme activity was determined by measuring the hydrolysis of substrate: N-Succinyl-L-Ala-L-Ala-L-Pro-L-Phe-P-nitroanilide (Suc-AAPF- pNA). Release of enzyme from Slaa gel, without prior soaking in TIDE^®, was also carried out in pH 7.85 buffer. The enzyme released by the gel system, over a period of 2 hours is given in Figure 2. The initial stock enzyme concentration was 34.5mg/ml; 100% release of enzyme would give an enzyme concentration of 34.5 mg/ml by the assay. The results showed that 80%> of enzyme (28 mg/ml) was released from Slaa gel in the pH 7.85 buffer, which indicates that the encapsulated enzyme is fairly intact in the gel matrix and there is no significant loss of enzyme activity during the gelation process. The enzyme activity value at time = 0 (1.05 mg/ml) indicates the amount of enzyme that leaked out after one day soaking in the detergent. The results show that 3% enzyme was leaked out into TIDE^® after the gel was soaked in TIDE^® for one day. The results also show that 50% enzyme was released in one hour after the gel-containing detergent is diluted in water. Example 4. Effect of additives to the Slaa gels Polysaccharides such as starch, pectin, sodium alginate and carrageenan (λ or type IV) were added to the silicate sol (Slaa gel). The enzyme-containing silicate sol was prepared according to Example 1. To the obtained sol, 0.5 ml of 2% aqueous solution of starch/pectin/sodium alginate/carrageenan (λ) was added, followed by the addition of 0.45 ml MTMOS sol. Translucent gels were obtained within 45 minutes. The gels obtained are referred as Slaa-starch, Slaa-pectin, Slaa-alginate and Slaa-Car(IV).

Example 5. Release of enzyme from Slaa - additive gels • (K) The enzyme containing gels were soaked for a day in TIDE detergent, which had been heated at 90°C for one hour to deactivate the enzymes contained therein. Then the release experiments were carried out by diluting the detergent that contains the gels into water. The enzyme released from different gel systems at different time intervals is given in Figure 3. The addition of polysaccharides appears to facilitate the enzyme release from the Slaa gels. The percentage of the enzyme released from the Slaa-additives gel systems after the detergent is diluted by water, are 10 minutes and 60 minutes are given in Figure 4. Addition of pectin appears to enhance the release of enzyme in the first 10 minutes.

While, pectin, alginate and carrageenan all seem to enhance the enzyme release in an hour after the dilution in water.

Example 6. Thermal Stability of protease in Sodium silicate gels MTMOS sol:

3 ml methyltrimethoxysilane + 2 ml H₂O + 0.033 ml HC1 (0.04 M) Sonication = 50 minutes

Sodium silicate solution: 0.75 ml Sodium silicate + 1.05 ml H₂O

Sodium silicate sol:

1.2 ml H₃PO₄ + 0.5 ml H2O + 1.8 ml sodium silicate solution + 0.45 ml MTMOS sol Gel composition:

Sodium silicate sol (0.3mL) + buffer (pH 5.6, 0.2mL) + enzyme (0.02mL) High temperature may cause shrinkage of the gel matrix. This in turn lowers the release of enzyme from the gel matrix. To minimize the influence of shrinkage of pores on enzyme release, the experiment has been designed where the substrate is added to the cuvette containing the gel with encapsulated enzyme. The idea is that the smaller substrate molecule would easily diffuse into the gel matrix and undergo hydrolysis. The rate of hydrolysis in the gel matrix is taken as the measure of the enzyme present in the gel. Sodium silicate gel containing the enzyme was formed on the wall of the cuvette. The gel was aged for a day before subjecting to a thermal treatment. The cuvette was filled with buffer (pH 5.5) and heated at 60°C in an oven for 4 h. A control experiment was also carried out where the gel with the same composition was left at room temperature under same conditions. After 4 h of heating, the buffer solution was changed from pH 5.5 to pH 8.6

(2mL) in the cuvette. The assay was carried out by adding 20 μl suc-AAPF-pNA substrate to the activity buffer contained in the cuvette.

Enzyme assays: a. Sodium silicate gel containing enzyme heated at 60°C for 4 h: = 0.2968 mg/mL b. Sodium silicate gel containing enzyme at room temperature (control): = 0.34 mg/mL

Considering the enzyme activity in the gel at room temperature as 100 %>, the activity of enzyme retained at 60°C = 0.2968 x 100 / 0.34 = 87.29%

For comparison, thermal stability of the unencapsulated stock enzyme was also carried out. A solution of stock protease in buffer (pH 5.6) was heated at 60°C for 4 h. Control experiment under room conditions was also carried out.

The enzyme assays obtained for the control experiments were - c. Stock enzyme + buffer (pH 5.6) heated at 60°C for 4 h: = 10.24 mg/ml d. Stock enzyme + buffer (pH 5.6) at room temperature = 35 mg/ml

The activity retained at 60°C in case of Stock enzyme in buffer (pH 5.6 ) = 10.14 x 100 / 35 = 29.26 % The enzyme encapsulated in silicate matrix retains 87%> of its activity on heating at

60°C for 4 h as compared to 29 % activity of the unencapsulated enzyme.

Example 7. Preparation of Sodium Meta Silicate gels Preparation of MTMOS sol To 6 mL of H₂O, 0.1 mL of HCl (0.04 M) and 9 mL of methyltrimethoxy silane were added and the mixture was sonicated for 45 minutes to obtain MTMOS sol.

Preparation of DMDS sol To 4mL of CH₃OH, 3mL of H₂O, 0.4 mL of HCl (0.04 M), followed by 1 mL of dimethyldimethoxysilane were added and sonicated for 45 minutes.

Preparation ofDMDS(l) sol To 3 mL of CH₃OH, 0.8 mL of H₂O, 0.4 mL of HCl (0.04 M), followed by 1 mL of dimethyldimethoxysilane were added and sonicated for 45 minutes.

MS(Metasilicate)la gel To a solution of 1.6 ml H₃PO₄ (3M), 0.85 ml aqueous sodium metasilicate solution

(1.3482 g / 3 ml H₂O) was added. To the sol, 0.75 ml stock enzyme was added. This was followed by addition of 0.45 ml MTMOS sol. Gel obtained was watery (not firm).

MS(Metasilicate) 1 ad gel To a solution of 1.6 ml H PO₄ (3M), 0.85 ml aqueous sodium metasilicate solution

(1.3482 g / 3 ml H₂O) was added. To the sol, 0.75 ml stock enzyme was added. This was followed by addition of 0.5 ml DMDS sol and 0.5 ml MTMOS sol. Gel obtained was opaque and firm.

MS(Metasilicate)ladl gel To a solution of 1.1 ml H₃PO₄ (3M), 0.9 ml aqueous sodium metasilicate solution

(1.3482 g / 3 ml H₂O) was added. To the sol, 0.75 ml stock enzyme was added. This was followed by addition of 0.5 ml DMDS (1) sol and 0.5 ml MTMOS sol. Gel obtained was opaque and firm.

Example 8. Release of enzyme from the metasilicate gels The release experiments were carried out as described for the sodium silicate gels (Example 3). Prior to use, TIDE^® detergent was heated at 90°C for one hour to deactivate the enzymes contained therein. The enzyme release experiment was carried out from (a) MS(metasilicate)la gel in pH 7.85 buffer without prior soaking in TIDE^®, (b) MS(metasilicate)la after soaking in TIDE^®, (c) MS(metasilicate)lad after soaking in TIDE^®, and (d) MS(metasilicate)ladl after soaking in TIDE . The results are shown in Figure 5. The results show that 86%> of enzyme was released from MS(metasilicate)la in pH 7.85 buffer (without soaking in TIDE ), which indicates the absence of loss of activity of the enzyme during the gelation process.

The %> enzyme release of the meta silicate systems is summarized in Table 2.

Table 2

(MS series were derived from sodium metasilicate sols) MSla gel showed 38% leakage of the enzyme from the matrix when the gel was soaking in the detergent for one day. MS lad gel, which was derived from DMDS sol along with the MTMOS sol, had a decrease in leakage to 27%. MSladl gel, which was derived from DMDS sol (higher amount than that of MS lad gel) along with the MTMOS sol, had a further decrease in leakage to 11%.

Example 9. Preparation of Silicate/siliconate (JA) gels A mixture of 1.0 mL of an aqueous sodium silicate solution (27%> w/w silica and 14%> w/w NaOH, Aldrich), 1.0 mL of an aqueous sodium methyl siliconate solution (30%> w/w, Gelest, NJ) and 2.0 mL H₂O was added with stirring to 2.0 mL H₃PO₄ solution (3M). The resulting mixture had a pH of about 6. To this solution was added 2 mL of stock protease (30 mg/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) with stirring. Gelation of the solution occurred within 30 minutes giving a clear yellowish gel.

Example 10. Preparation of Sodium Silicate Gels encapsulating a Dye Silicate 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. Preparation of MTMOS sol. To 6 mL of H₂O, 0.1 mL of HCl (0.04 M) and 9 mL of

Methyltrimethoxysilane were added and the mixture was sonicated for 45 minutes to obtain MTMOS sol. Silicate sol. To a solution of 1.2 ml H₃PO₄ (3M) and 0.5 ml H₂O, aqueous sodium silicate solution containing 0.75 ml sodium silicate solution (27%_> w/w silica and 14% w/w NaOH, Aldrich, WI) and 1.05 ml H₂O was added to form a silicate sol. To the silicate sol, 0.75 ml of 0.02 mg/mL Chicago Sky Blue dye (in water) was added, followed by 0.5 mL of 2%> aqueous solution of starch. This was^" followed by addition of 0.45 ml MTMOS sol. All the components were added dropwise while stirring the sols continuously. Gels were formed in about 45 minutes. The obtained gels were blue translucent gels with a firm appearance. When 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 11. Enzyme leakage during storage in detergent and enzyme release upon dilution in water. Silicate/siliconate sol-gels (JA) were prepared according to Example 9. Silicate- starch gels (Slaa-starch or SS) were prepared according to Example 4 where the starch used was PURE-COTE^® B790 (Grain Processing Corp., Muscatine, LA). Prior to use, TIDE^® detergent was heated at 90°C for one hour to deactivate the enzymes contained therein. Each silicate/siliconate (JA) and silicate/starch gel (SS) monolith was immersed in a TIDE^® and stored for 1-4 weeks. There was no rotation during storage. At the end of each storage period, the samples were rotated at 8 rpm 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 % enzyme released from the gels into detergent during static storage in detergent was calculated. Both the JA and SS gels gave similar results. After 1, 2, 3, and 4 weeks storage in the detergent, a total of 5, 10, 18, and 19% of enzyme was leaked out of the gels respectively. After storage in the detergent for 1, 2, 3, or 4 weeks, the gels were also tested for their responsiveness to dilution in water. Each gel-containing detergent formulation was diluted 10-fold in water and agitated at 8 rpm for about an hour. For the JA gels, the extent of enzyme released upon dilution was observed to increase after longer storage in the detergent. For the SS gels, the extent of release was mostly independent of storage time in the detergent. The results are summarized in Figure 6. 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. A method for preparing enzyme-containing gels comprising the steps of: (a) mixing a first aqueous solution with one or more alkali metal silicates or alkyl siliconate salts to lower the pH to 12 or lower to form a first sol; (b) mixing a second aqueous solution with one or more organofunctional silane precursors selected from the group consisting dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, aminoalkylsilane, 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; wherein the one or more enzymes are contained within the gels and are released from the gels when the gels are placed in a solution having water percentage by weight of 86%> or more.

A method for preparing enzyme-containing gels comprising the steps of: (a) mixing (i) a first aqueous solution, (ii) one or more alkali metal silicates or alkyl siliconate salts, and (iii) one or more organofunctional silane precursors selected from the group consisting of dialkyldialkoxysilane, alkyltrialkoxysilane, alkylsilane, aminoalkylsilane, to form sols; (b) mixing the sols with a second aqueous solution containing one or more enzymes; and (c) incubating (b) to form gels; wherein the one or more enzymes are contained within the gels and are released from the gels when the gels are placed in a solution having water percentage by weight of 86%> or more.

3. The method according to Claim 1 or 2, wherein said dialkyldialkoxysilane is dimethyldimethoxysilane.

4. The method according to Claim 1 or 2, wherein said alkyltrialkoxysilane is methyltrimethoxysilane.

5. The method according to Claim 1 or 2, wherein said aminoalkylsilane is aminoalkyltrialkoxysilane, bis(trialkoxyalkylsilane)alkylenediamine, bis(dialkyldialkoxysilane)amine, bis(trialkoxyalkylsilane)amine, and aminoalkylsilanetriol or its salt.

6. The method according to Claim 5, wherein said bis(trialkoxyalkylsilane)alkylenediamine is bis[(trimethoxysilyl)propyl]ethylenediamine.

7. The method according to Claim 5, wherein said bis(trialkoxyalkylsilane)amine is bis[3-trimethoxysilyl)-propyl]amine.

8. The method according to Claim 5, wherein said bis(dialkyldialkoxysilane)amine is bis(methyldiethoxysilylpropyl)amine.

9. The method according to Claim 5, wherein said aminoalkyltrialkoxysilane is 3- aminopropyltrimethoxysilane.

10. Te method according to Claim 1 or 2, wherein said alkylsilanetriol is aminopropylsilanetriol.

11. The method according to Claim 1 or 2, wherein said alkali metal silicate is sodium silicate or potassium silicate.

12. The method according to Claim 1 or 2, wherein said alkyl siliconate salt is sodium alkyl siliconate or potassium alkyl siliconate.

13. The method according to Claim 1, further comprising the steps of hydro lyzing negatively charged organosilane precursors to form a third sol, and mixing the third sol with the first sol, the second sol, and the aqueous solution containing one or more enzymes.

14. The method according to Claim 2, wherein step (a) further comprises mixing (iv) one or more negatively charged organosilane precursors.

15. The method according to Claim 13 or 14, wherein the negatively charged organosilane precursors are trialkoxysilylalkyl succinic anhydride, trihydroxysilylalkylphosphonate, trihydroxysilylalkylsulfonic acid, or carboxyalkylsilanetriol.

16. The method according to Claim 15, wherein the negatively charged organosilane precursors are 3-(triethoxysilyl)propyl succinic anhydride, 3-trihydroxysilylpropylmethyl phosphonate, 3-trialkoxysilylpropylmethyl phosphonate, 3-(trihydroxysilyl)-l- propanesulfonic acid, carboxyalkylsilanetriol, or carboxyalkyltrialkoxysilane.

17. The method according to Claim 1, wherein one or more disaccharides, polysaccharides, polyvinyl alcohols, or polyethylene glycol are added to step (a), (b) or (c).

18. The method according to Claim 2, wherein one or more disaccharides, polysaccharides, polyvinyl alcohols, polyethylene glycol, or polypropylene glycol are added to step (a) or (b).

19. The method according to Claim 17 or 18, wherein said one or more polysaccharides are starch, pectin, sodium alginate, or carrageenan.

20. The enzyme-containing gels prepared according to the method of Claim 1 or 2

21. The enzyme-containing gels according to Claim 20, wherein said one or more enzymes are encapsulated or embedded within the gels.

22. The enzyme-containing gels according to Claim 20, wherein said one or more enzymes are thermally stable.

23. The enzyme-containing gels according to Claim 20, wherein at least 30%_> of said one or more enzymes are released from the gels within an hour upon diluting the gels into a solution that increases the water percentage by weight to 86% or higher.

24. A liquid detergent formulation comprising the enzyme-containing gels according to Claim 20.

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

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

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

28. The liquid detergent formulation according to Claim 24, wherein said one or more enzymes are detergent enzymes used in laundry detergents, fabric care products, or dishwasher detergents.

29. The enzyme-containing gels according to Claim 28, 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.

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