WO2004078773A1 - Stabilizing proteins for use in personal care, cosmetic, and pharmaceutical products - Google Patents

Abstract

Several secreted proteases (Figure 5) are produced with dedicated single-turnover intramolecular chaperones (IMC) or propeptides. Protease maturation necessitates IMCs to switch from a chaperone to a protease inhibitor and subsequently into a proteolytic substrate. The present invention provides compositions and methods using environmental signals to modulate the stochastic behavior associated with the inhibitor-substrate switch between infinitely probabilistic and precisely deterministic temporal events. This provides a unique biological switch that enable proteases to function as biochemical regulators through modulation of conformational entropy of their cognate IMCs. The present invention provides compositions comprising proteases that are stabilized as inactive proteases, but that can yet be rapidly activated by external signals (Figure 4).

Description

STABILIZING PROTEINS FOR USE IN PERSONAL CARE, COSMETIC, AND PHARMACEUTICAL PRODUCTS

CROSS REFERENCE TO RELATED APPLICATIONS This a pplication c laims the b enefϊt o f p riority t o U nited S tates P ro visional P atent

Application Number 60/451,883, filed 03 March 2003, entitled Stabilizing Proteins For Use In Personal Care, Cosmetic, Pharmaceutical Products, and incorporated by reference herein.

SPONSORED RESEARCH OR DEVELOPMENT This invention was supported by a Medical Research F oundation of Oregon Seed

Grant, December 2001-2002.

BACKGROUND OF THE INVENTION The biological activity of proteins is dependent on the proteins assuming their proper three-dimensional conformation. The proper conformation determines the activity and/or stability of the proteins. Most proteins possess, within the sequence of the polypeptide itself, all the information necessary for folding into the active conformation. Proteins are typically denatured by subjecting them to temperatures above or below the physiological temperature range, or to denaturing agents or chemicals, such as urea or guanidine hydrochloride.

Particular proteins, self-refolding proteins, are capable of refolding to their original biochemically active conformation upon equilibration to physiological temperature, or upon removal of the denaturant. However, other proteins (e.g., subtilisin, α-lytic proteases, and carboxypeptidase) are unable to effectively self-refold once they are denatured, even when the proteins are removed from the denaturing condition or agent. Within the group of proteins which cannot self-refold properly into a biochemically active conformation, pro- subtilisin, a serine protease, has been established as a model for analyzing protein-folding mechanisms (see, e.g., U.S. Patent No. 5,719,021 hereby incorporated by reference in its entirety). Proteases such as subtilisins have found much utility in industry, particularly in detergent formulations, where they are useful for removing proteinaceous stains. For example, the following proteases are known and marketed in large quantities in many countries of the world: Subtilisin BPN¹ or Novo, available from e.g. SIGMA, St. Louis, U.S.A.; Subtilisin Carlsberg, marketed by Novo-Nordisk A/S (Denmark) as ALCALASE™ and by IBIS (Holland) as MAXATASE™; a bacillus lentus subtilisin, marketed by NOVO LNDUSTRI A/S (Denmark) as SAVTNASE™; enzymes closely resembling SAVLNASE™ such as MAXACAL™ marketed by IBIS and OPTICLEAN™ marketed by MILES KALI CHEMIE (FRG); a bacillus lentus subtilisin, marketed by Novo Nordisk A/S (Denmark) as ESPERASE™; and KAZUSASE™ marketed by SHOWA DENKO (Japan). Other examples are available in, for example, U.S. Patent No. 6,521,577, incorporated herein by reference in its entirety.

During the past few decades various types of proteases from plants and microbial sources have become popular in the pharmaceutical area as so-called debridement agents, hi this application proteases like collagenase, subtilisin and papain are used to clean infected wounds and to destroy necrotic tissue in cases of decubitus or severe burns.

Many enzymes are catalysts and responsible for the basic chemical reactions required for maintaining and propagating life. Approximately 2,500 different enzymes are known in the scientific literature but less than 100 enzymes are commercially available. Enzymes are protein chains folded in a characteristic three-dimensional structure. This three-dimensional structure is essential for the enzyme's catalytic activity, and the respective reactions typically proceed at ambient temperature and near neutral pH conditions, rather than at pH and temperature extremes where the three-dimensional structure is lost.

Most enzymes are unstable and have poor shelf stability because their three- dimensional structure is gradually lost in the presence of water. When this three- dimensional structure is lost, the enzyme becomes inactive and constitutes 'dead' protein. Examples of the use of such ^εdead' proteins in cosmetics are hydrolyzed soya, gluten or silk proteins which are used in large quantities to repair damaged skin and hair.

By inference, most enzymes will be relatively stable in the absence of water or under conditions of low water activity/concentration. For example, freeze-drying enzyme solutions typically yields a relatively stable enzyme powder. Additionally, high percentages of compounds like glycerol or butylene glycol are known to lower water-activity, thereby yielding relatively stable enzyme solutions. However, while these enzymes may be stable, they are not active in such dry or stabilized states. Enzymes typically require water to be active, but the presence of water slowly inactivates the enzyme. Therefore, most enzymes incorporated into, for example, a cosmetic product with a high water activity will lose all of their activity within a few weeks. Enzymes incorporated into a cosmetic product with a low water-activity a re s table b ut a re, u nfortunately, inactive o n t he d ry s kin o r w hen u sed i n other manners. Because of the small molecular weight of water, the current method of effective stabilization and activation of enzymes requires a robust or stringent separation of enzyme and water (e.g., a two-chamber packaging concept), and two-chamber dispensers for cosmetic applications have been recently introduced. In these packages, one chamber contains the enzyme in a stabilized form, for example, as a dry powder or an enzyme concentrate dissolved in glycerol, while the other chamber contains the water, required to activate the enzyme, incorporated in the cosmetic cream or lotion. However, such two- chamber dispensers are relatively expensive, complicated, and prone to more difficulties when compared to conventional tubes or dispensers. The one billion market for cosmetics and personal care is a relatively new area for enzyme applications, and enzyme stability and activation are major impediments to the effective application of enzymes in cosmetics, personal care products, and pharmaceuticals. Enzymes are employed in a number of products, including, but not limited to the use of: proteases, Upases and catalases for contact lens c leaners; and g lucoamylases and glucose oxidases in toothpastes and proteases in denture cleaners. Additionally, enzymes, and proteases in particular, have become popular in cosmetics to clean and smoothen the skin.

Practical problems to the effective application of enzymes for cosmetics and personal care products include, but are not limited to, unfavorable side effects, inadequate regulation of activity, and instability. Before enzymes can be applied in cosmetics, the poor shelf stability of enzymes in cosmetic formulations must be resolved.

Likewise, the formulation of enzyme-containing aqueous liquid detergent compositions i s v ery d ifficult d ue t o t he r apid d ecrease i n e nzymatic a ctivity i n a queous detergent media during storage. Particular additives have been added to formulations containing enzymes in an attempt to stabilize those enzymes (see, e.g., U.S. P atent Nos. 4,261,868, 4,404,115, 4,318,818, 4.243,543, 4,462,922, 4,532,064, and 4,537,707, incorporated herein by reference in their entireties.

Certain enzymes are of particular interest to the cosmetic industry, such as those enzymes that are exceptionally stable even in the presence of water (e.g., superoxide dismutase (SOD) and lactoperoxidase). Lactoperoxidase is an enzyme present in a.o. saliva and milk and able to form natural biocidal compounds. P resently, this enzyme i s b eing commercialized as a preservation system for cosmetic creams.

More recently cosmetics incorporating relatively unstable enzymes have been launched. Such cosmetics contain either lipoxygenase, protease or lipase. Lipoxygenase is a highly unstable enzyme with a strong depilatory effect. Proteases are of mediocre stability but of interest because they are able to smoothen and soften the skin by releasing squamae from the epidermis. Not surprisingly, cosmetics incorporating unstable enzymes are currently being marketed in two-chamber dispensers.

Besides enzymes for contact lens and denture cleaners, the personal care area focuses on enzymes for skin, hair and dental care. Customers in other industries (e.g., the wine and juice, alcohol, brewing, pulp and paper, and leather industries) are also likely to benefit from t he m any new p roducts t hat are c urrently u nder d evelopment, a s i t i s o ften possible to transfer applications of enzymes between the different industries.

Additionally, two-thirds of the market for technical enzymes is made up by detergent additives. Major detergent enzymes are proteases, lipases, amylases and cellulases. These enzymes are used as functional ingredients in laundry detergents and automated dishwashing detergents. One major obstacle to be overcome in this context is enzyme instability.

The proteinaceous soil-removing capabilities of detergent compositions are believed to be very significant in today's marketplace. Except under unusual conditions, other types of stains, for example stains comprising carbohydrate or lipophilic materials, can be efficiently removed by other means (e.g., anionic or nonionic detergents). Proteases, are believed to provide a significant contribution to the efficiency of a liquid detergent composition, particularly in laundering and foam-and-clean applications. Detergent manufacturers can no longer rely as heavily upon phosphate-containing detergents and hot water washing techniques, because there is pressure to reduce the level of phosphates from an environmental standpoint, and lower wash water temperatures to help conserve energy. The net effect is that many modern detergents and modem washing techniques are actually less efficient in removing certain types of stains. Furthermore, recourse to bleaching agents may be undesirable with respect to certain colors or types of fabrics. Proteolytic enzymes increase cleaning efficiency, even in low temperature washing. Furthermore, certain proteinaceous stains such as blood stains may actually be more difficult to remove with hot wash water. The hot water tends to denature the blood protein and thus "set" the stain, making it more difficult to solubilize. Beyond cosmetological and industrial applications, disease conditions, such as

Alzheimer's disease, Creutzfeldt- Jakob disease, cystic fibrosis and p53-related cancers are all associated with incorrect protein folding, and are major causes of morbidity, mortality and healthcare costs. To ensure that proteins fold correctly, biological systems have evolved elaborate checkpoints that utilize chaperones and proteases. Despite such checkpoints, proteins can misfold and cause serious damage to the host organism. While it is widely accepted that protein misfolding leads to loss or gain of function, the mechanisms that promote these altered functions have not been understood. A lack of understanding of these issues represents a major problem because it hinders the prevention of incorrect folding and impedes the development of tailor-made protein folding catalysts. There is an accepted theory that proteins fold through fixed pathways and the discovery of folding intermediates is consistent with this theory. Recent experiments have also established that unique amino acid sequences can acquire multiple active conformations while two non- homologous proteins can adopt similar folds. However, it is likely that every folding problem does not have a unique solution.

Thus, there is a need in the art to identify mechanisms of protein misfolding and to design peptides that direct efficient protein folding There is a need to: understand the significance of folding intermediates and the role of their environment on folding; identify common structural determinants that initiate protein folding; and design denovo peptide chaperones.

There is a need in the art to improve enzyme stability for cosmetological and industrial applications. There is a need in the art to more effectively retain enzymes in inactive and/or stable configurations. There is a need in the art to activate enzymes in a facile and controlled manner. These and other limitations and problems of the past are solved by the present invention.

BRIEF SUMMARY OF THE INVENTION Intramolecular chaperones (LMCs) are N-terminal propeptides that function as single-turnover catalysts that guide certain proteases from unfolded states to meta-stable native states through thermodynamically stable inhibition complexes. Single-turnover separates the folding and unfolding pathways, and folded proteases acquire stability because of high unfolding barriers between the kinetically trapped meta-stable conformations and unfolded states. This enables proteases to be stable in harsh environments. Proteases can also function as regulatory molecules and this requires their activation be stringently controlled on a spatial and temporal level. The consequence of such stability has forced a class of proteases to evolve dedicated intramolecular chaperone-domains that chaperone the enzymatic-domain into kinetically trapped metastable conformations through thermodynamically stable inhibition complexes. After chaperone-mediated folding, the LMC-domain is cleaved at its primary cleavage site to stabilize the inhibition complex.

The release and the second cleavage of the now inhibitory I MC-domain from the cleaved complex represent the rate-determining step (RDS) in precursor maturation. The first free protease molecule thus formed during the RDS, can 'feed back' to degrade the LMC that is tightly associated with another protease molecule through trans proteolysis. Hence, the LMC switches from a chaperone to a protease inhibitor and subsequently into a proteolytic substrate on different time scales and in a coupled non-linear manner. The RDS step may causes protease activation to be under stochastic control and the coupled feed-back network provides a mechanism to regulate the protease activation precision. Particular aspects of the present invention are directed to stabilizing proteins through the action of intramolecular chaperones (LMCs). hi particular, the present invention is directed to maintaining proteases in an inactive state, which can then be activated upon demand through an external signal. These activated proteases have usefulness in cosmetic, personal care, industrial and pharmaceutical compositions. Further aspects of the invention relate to compositions containing proteolytic enzymes, wherein the proteolytic activity of the enzyme has been generally stabilized against deterioration (e.g., denaturization or degradation of the enzyme molecule).

In particular embodiments, the present invention provides a protease maintained in an inactive state that can be activated upon demand through an external signal. Preferably, the protease is member of the subtilisin family.

Additional embodiments provide compositions containing one or more proteases maintained in an inactive state that can be activated upon demand through an external signal.

Yet further embodiments provide methods of obtaining a protease in an inactive state capable of activation by a signal.

In particular aspects, the invention provides an activatable protease-containing composition, comprising at least one of a pro-subtilisin protease comprising an N-terminal propeptide that is an intramolecular chaperone (IMC), and a subtilisin protease that is non- covalently 1 oaded w ith a n N -terminal p ropeptide s uitable a s a n i ntramolecular c haperone (LMC), wherein the proteases are inactive, or substantially so; a stabilizing agent; and a sequestered activator sufficient to rapidly activate the inactive protease upon triggered release of the activator. Preferably, the stabililzing agent is selected from the group consisting of from about 5% to about 25% glycerol, (NH₄)₂SO₄ from about 0.5 to about 1.5 M, and combinations thereof. Preferably, the activator, in the non-sequestered state, is selected from the group consisting of SDS at about 0.005% to about 0.05%, active subtilisin from about 1 to about 10 nM, an agent that establishes a pH value between about 7.5 and about 10.00, and dilution, in the case of an (NH₄)₂SO₄ stabilizer, of the (NH₄)₂SO₄ concentration to a value at or below about 0.4 M. In additional aspects, the invention provides A method of activating, upon demand, an inactive subtilisin protease, comprising: obtaining a composition having at least one of a pro-subtilisin protease comprising an N-terminal propeptide that is an intramolecular chaperone (LMC), and a subtilisin protease that is non-covalently loaded with an N- terminal propeptide suitable as an intramolecular chaperone (LMC), wherein the proteases are inactive, or substantially so, and wherein the composition also comprises a stabilizing agent, and a sequestered activator sufficient to rapidly activate the inactive protease upon triggered release of the activator; and triggering the activator.

Yet further aspects of the invention provide a method of manufacturing activatable protease-containing compositions having a prolonged protease shelf-life, comprising: obtaining a composition comprising at least one of a pro-subtilisin protease comprising an N-terminal propeptide that is an intramolecular chaperone (IMC), and a subtilisin protease that is non-covalently loaded with an N-terminal propeptide suitable as an intramolecular chaperone (LMC), wherein the proteases are inactive, or substantially so; adding a stabilizing agent thereto; and adding thereto a sequestered activator sufficient to rapidly activate the inactive protease upon triggered release of the activator, whereby the protease shelf-life is prolonged in a state that is activatable upon demand by triggered release of the sequestered activator.

The invention will best be understood by reference to the following detailed description of the preferred embodiments, taken in conjunction with the accompanying drawings. The discussion below is descriptive, illustrative and exemplary and is not to be taken as limiting the scope defined by any appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figures la-le show stochastic activation of pro-subtilisin. Figure la shows a schematic representation of prosubtilisin maturation with the rate-determining step and trans autocatalytic activation. Figure lb shows stochastic activation of multiple aliquots from a single folding reaction captured at different time intervals using a chromogenic substrate. Figure lc shows enzyme activity as a function of time, for 12 randomly selected samples from a maturation reaction. Inset depicts SDS-PAGE of pro-subtilisin maturation with active (+) and inactive (-) aliquots. Figure Id shows frequency distribution of the number of active aliquots in a microplate as a function of time. Figure le shows subtilisin yield (open circles) and average activation time (filled squares) as a function of precursor concentration. Figures 2a-c show signal-induced deterministic activation. Figure 2a shows deterministic activation induced by external signals. The leftmost panel depicts control for stochastic activation while the other panels represent signal-induced activation at different times. Figure 2b shows the effect of signals monitored using SDS-PAGE. Lanes 7-10 represents maturation of prosubtilisin control, while lane 1 depicts the starting material. Addition of signals (subtilisimlane 4; Tris-HCl:lane 5; SDS:lane 6) lhr after folding initiation induces degradation of precursor and LMC-domains. Lane 3 represents the amount o f s ubtilisin a dded a s a s ignal t o 1 ane 4. F igure 2 c s hows s ubtilisin yield w hen signals are added at different times.

Figures 3a-e show signal-induced conformational stabilization. Figure 3 a shows frequency distribution of the number of active aliquots (black bars) as a function of time, under normal and glycerol conditions. Signal instantaneously activates all wells (Red bar). Figure 3 b shows the effect of stabilizing signals monitored using SDS-PAGE (lanes 2- 5:2hr and lanes 7-10:8hr samples). Only control autodegrades its LMC-domain in the absence of signal (Lane 7, 8). Figure 3c shows the averaged activation under normal and stabilized conditions (glycerol, salt and glycerol-salt). Un-induced (dotted lines) and induced (solid lines) activation due to signals (0.01 % SDS) added at times indicated. Figure 3d shows circular dichroism spectra for inhibition complex (broken line), mature (solid line) and IMC-domain, free (dotted line) and in complex (thick broken line) under normal (blue) and stabilized (red) conditions. The spectra for the LMC-domain in complex were o btained from t he difference b etween t he i nhibition c omplex a nd m ature s ubtilisin. Figure 3e shows proteolytic stability of the LMC-domain in the inhibition complex under normal (blue) and stabilized (red) conditions against TPCK-treated trypsin.

Figures 4a-c show the energy landscape for protease maturation. Figure 4a shows Arrhenius plots for activation rate. Figure 4b shows slow binding inhibition of subtilisin by its LMC-domain. Time-dependent activity of subtilisin was measured using the LMC concentrations as indicated. Figure 4c shows a free energy diagram of protease activation. Activation requires transition of the protease from its thermodynamically stable inhibition complex to a kinetically trapped active form. Stochastic behavior occurs due to the nonspontaneous nature of the process that relies on a probabilistic rate determining step (RDS), namely release and degradation of the inhibitory LMC-domain.

Figure 5 shows a primary sequence alignment between the subtilisin propeptide (ProWT) and the redesigned propeptide (ProD) (taken from Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003). Identical residues (black background) and highly conserved substitutions (gray background) and less conserved substitutions (bold-type faced) have been previously identified (Id). The secondary structure of ProWT (X-ray structure) when it forms a complex with subtilisin is shown depicted above the sequence alignment, while the computed secondary structure is depicted below the sequence alignment. The letters C, E and H denote coils, β-sheets and α-helices, respectively, while the predicted structure in the bold-type faced represents predictions that do not match with the secondary structure obtained from the crystallized complex of ProWT: subtilisin. Motifs NI and N2 represent the conserved domains within the subtilase family. The asterisks denote residues that constitute the hydrophobic core within the propeptide.

DETAILED DESCRIPTION OF THE INVENTION

Certain proteases are produced as precursors, with propeptide extensions that function as dedicated single-turnover protein folding catalysts. Propeptides are termed 'intramolecular chaperones' (LMC), because they are covalently attached to the catalytic domain, and they help to properly fold the protein. Single turnover is a consequence of the structural and proteolytic instability of the LMC.

Described herein are novel compositions, and methods for modulating the structural and proteolytic stability of proteases (e.g., subtilisins) through changes in the environment surrounding the precursor (e.g., prosubtilisin), so as to (i) be able to stabilize the protease in a catalytically inactive state, and (ii) to allow for rapid (and in some instances, almost instantaneous) activation of the protease with a small external environmental signal. This novel approach allows the protease to be kept inactive, thereby prolonging the life of the protease. Once activated, protease compositions can undergo autolysis, and therefore such composition have a shorter shelf life.

According to aspects of the present invention, stabilized proteases having an extended shelf-life, and which can be readily activated by external signals, are ideal for use in, ter alia, cosmetics, personal care and pharmaceutical products.

Protease activity and source. The activity of proteolytic enzymes is usually expressed in art-recognized Anson units, or glycine units (1 Anson unit/g=733 glycine units/mg). Compositions of the present invention include a proteolytically effective amount of a proteolytic enzyme, a class of enzymes generally referred to as proteases. Proteolytic enzymes have a protein-like or polypeptide structure made up of repeating amino acid units; that is, the -COOH (carboxyl) function of a first amino acid unit combines with an -NH₃ (amino) function of a second amino acid unit to form the peptide linkage which is in essence an amide linkage. Typical proteolytic enzymes contain tyrosine and/or tryptophan and/or methionine and/or histidine units, all of which are sensitive to oxidation. Much of the loss of proteolytic enzyme activity when the enzyme is distributed through a water phase is believed to stem from degradation or attack upon these units, which attack is essentially an oxidation process or is accelerated by the presence of air or oxygen. Autolysis i s a lso a c ause o f d egradation. O ther t ypes o f enzymes u seful for t he p resent purposes contain cysteine residues. Cysteine-containing proteins may contain disulfide (— S--S--) bonds, which are also sensitive to oxidative/reductive modification.

An aspect of the proteolytic enzymes utilized in the present invention is pH sensitivity. Many of these enzymes lose a substantial amount of activity or otherwise become less effective at a pH below 5.2 or above 9.0; adverse effects upon proteolytic enzyme activity can be observed at a pH as low as 5.5 or as high as 8.0.

Proteases are generally considered to include enzymes which hydrolyze peptide linkages, regardless of whether the peptide linkage is part of a low molecular weight polypeptide (e.g. a polypeptide containing only a few amino acid units) or part of that class of polypeptides referred to as proteins, which typically contain more than about 100 amino acid units and have molecular weights in the thousands, tens of thousands, or hundreds of thousands. Most typically, of course, the enzymes are selected on the basis of their ability to attack p roteinaceous stains, s uch a s b lood s tains, m ilk s tains, c ocoa s tains, o ther food stains, and certain types of stains from vegetable matter (e.g. grass stains). The products of the attack upon the proteinaceous stains can be individual amino acid units, relatively low molecular weight polypeptides, or both.

The proteolytic enzymes of the present invention can be of animal, vegetable or microorganism origin. Vegetable sources for proteolytic enzymes are known, e.g. papaya, pineapples, and the like; papain being typical of such proteases from vegetable sources. However, the more common practice from a commercial standpoint is to make large quantities of proteolytic enzymes from spore-forming organisms such as Bacillus species and Bacillus subtilis. Typical disclosures of commercially available proteases are contained in U.S. Patent No. 3,627,688 and U.S. Patent No. 3,746,649, both incorporated by reference herein. The proteases for use in detergent composition embodiments herein include, but are not limited to, trypsin, subtilisin, chymotrypsin and elastase-type proteases. Further examples of some commercially available proteases are found in U.S. Patent No. 6,521,577 (incorporated herein in its entirety). Preferably, the protease is a member of the subtilisin family.

Applications. The growth of the contact lens industry has led to a dramatic increase in the number of lenses and respective care regimens in the marketplace. Designing care regimens to meet the needs of all possible permutations is a challenge to the industry. Significantly, a goal of the lens-care industry is to simplify the lens care regimen to obtain greater patient compliance.

In the normal course of wearing contact lenses, tear film and debris consisting of proteinaceous, oily, sebaceous, and related organic matter, have a tendency to deposit and build up on lens surfaces. As part of a routine care regimen, contact lenses must be cleaned to remove these tear film deposits and debris, and if these deposits are not properly removed, both the wetability and optical clarity of the lenses is substantially reduced, causing discomfort for the wearer, and possible infections, etc.

Currently, the only safe and effective means for removing protein build-up is the use of enzymes, the hydrolytic activity of which reduces the proteinaceous materials to small, water-soluble subunits. Particularly useful are proteolytic enzymes or proteases. U.S. Patent No. 3,910,296, for example, discloses the use of proteases for cleaning contact lenses.

Prior art liquid enzyme compositions comprise, for example, an enzyme, stabilizing agents and water. Prior art stabilizing agents include, for example, monomeric polyol, a polymeric polyol, calcium ion and a borate/boric acid compound as described in U.S. Patent No. 6,214,596.

In one embodiment, personal care, cosmetic, therapeutic, and pharmaceutical compositions can be prepared using a protease stabilized according to the present invention. The stabilized protease is capable of being dissolved or suspended in suitable carriers, such as but not limited to, vegetable oils, such as seed oil or soy-bean oil, lecithin, glycerol, glycerylfurfurole, Tween 80 or other derivatives, suspending agents or diluents. The cosmetic, therapeutic, and pharmaceutical compositions for topical application can be formulated as pastes, creams, ointments, gels, lotions, cleaning solutions, and/or unguents or the like. Some products that may benefit from the inclusion of proteases with prolonged shelf life, as discussed herein, are denture cleaners, wound cleaners, liquid detergents, powder detergents, soap bars, skin care lotions, contact lens cleaners, liquid soaps, hard surface cleaners, and dishwashing compositions, among others.

As described in U.S. Patent No. 6,514,926, enzymes are normally incorporated into detergent or detergent additive compositions at levels sufficient to provide a "cleaning- effective amount." The term "cleaning effective amount" refers to any amount capable of producing a cleaning, stain removal, whitening, deodorizing, or freshness improving effect on substrates such as fabrics, dishware and the like. Typical amounts for current commercial p reparations are up to about 5 mg by weight, more typically 0.01 mg to 3 mg., of active enzyme per gram of the detergent composition. Stated otherwise, the compositions herein will typically comprise from about 0.001% to about 5%, and preferably about 0.01% to about 1% by weight of a commercial enzyme preparation. Protease enzymes are usually present in such commercial preparations at levels sufficient to provide from about 0.005 to about 0.1 Anson units (AU) of activity per gram of composition. For certain detergents it may be desirable to increase the active enzyme content of the commercial preparation.

Inventive detergent compositions, as described in the examples herein, include one or more enzymes which provide cleaning performance benefits. Said enzymes include enzymes selected from cellulases, hemicelluloses, peroxidases, proteases, gmco-amylases, amylases, lipases, cutinases, pectinases, xylanases, reductases, ocidases, phenoloxidases, lipoxygenases, hgninases, puUulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases or mixtures thereof, h one aspect, a detergent includes a cocktail of applicable enzymes like proteases, amylase, lipase, cutinase and/or cellulose. These enzymes may be in an inactive, stable state, capable of being activated by one of the 'external' signals described under Example 3 herein. Although subtilisin, an alkaline serine protease is used as an example herein, the method and composition herein can be used with several other related proteases.

Stochastic behavior associated with protease activation. To establish the stochastics

(random variables) associated with protease activation, prosubtilisin folding was initiated at room temperature, at or about 23°C, through a rapid-dilution technique (Fu, X.F., et al., J.

Biol. Chem. 275: 16871-16878, 2000), as detailed in the Examples below. The emergence of enzyme activity in different aliquots was monitored.

Fig. lb and Fig. lc establish that individual aliquots undergo 'none-to-all' activation at different times, although they represent the same folding reaction. As shown in Fig. Id, the plot of number of wells active versus activation time displays a Poisson's distribution with a mean of 235 minutes. While stochastic activation was observed at different precursor concentrations, Fig. le demonstrates that the time of activation increases exponentially at low precursor concentrations (<50 nM) and is consistent with an auto- catalyzed bimolecular (transproteolysis) reaction. At higher precursor concentrations activation time is limited by turnover of the inhibitory LMC-domain.

Example 2 shows the amount of active subtilisin estimated in individual wells of the microplate when folding was carried out using different precursor concentrations. As shown in Fig. le, the yield of protease varied less than 1 percent between individual aliquots and was linearly dependent upon the precursor concentration used for folding. The data indicates that protease activation displays a stochastic behavior that does not arise due to aggregation, because precipitation would be expected to diminish yields of active protein. Lack of aggregation was further confirmed using gel filtration chromatography.

To analyze differences between enzymatically active and inactive aliquots, pro- subtilisin maturation was terminated by trichloroacetic acid (TCA) precipitation, after 20- 30% of the wells displayed protease activity. An SDS-PAGE analysis established that while the amount of mature protease is similar (±1 %), the inhibitory LMC-domain is absent from enzymatically active aliquots as shown in Fig. lc, inset. This suggests that the observed stochastic behavior is due to differences in rates of degradation of the inhibitory LMC-domain.

Signal induced deterministic activation. As shown in Fig. 2a, additives or external 'signals' were identified that expedite precursor activation as described Example 3. The efficacy of each signal is different, with 0.01% SDS being the most effective, causing essentially instantaneous activation (Fig. 2a). Moreover, all signals promote specific degradation of the inhibitory precursor and cleaved LMC-domain, as shown in Fig. 2b.

When activation was expedited by adding signals at different times after the completion of the first cleavage, the yield of mature subtilisin was found to be independent of both the types of signals and the time they were added, as shown in Fig. 2c. This confirms that precursor maturation is complete within 30 minutes of folding initiation and the cleaved complex is in an active competent state. Therefore, the prolonged inhibition is not required to increase protease yield by preventing premature proteolysis.

Signal induced stabilization of stochastic behavior. Since certain signals can expedite activation, signals that prolong (i.e., delay) precursor activation w ere i dentified. The isolated LMC-domain is unstructured but adopts significant secondary structure in glycerol and trifluoroethanol. (Shinde, U.P., et al., Proc. Natl. Acad. Sci. USA 90:6924- 6928, 1993; Bryan, P. et al., Biochemistry 34:10310-10318, 1995; and Eder, J. et al., J. Mol. Biol. 233: 293-304,1993). Furthermore, enhancing the independent stability of the lMC- domain increases its affinity for its cognate protease. (Ruan, B., et al., Biochemistry 38: 8562-8571, 1999; Kojima, S. et al., J Mol. Biol. 277:1007-1013, 1998).

Solvents were examined to determine whether they could induce structure within the LMC-domain prolonging release of active protease, while still allowing for rapid activation in response to an external signal. Fig. 3a shows data demonstrating that while the presence of glycerol (e.g., 10%) increases the activation time and stochastic distribution by approximately three-fold, addition of SDS-signal instantaneously activates the complex under both normal and stabilizing conditions by expediting LMC-degradation (Fig. 3b).

Also identified herein are high salt and a high salt-glycerol combinations as stabilizers that prolong (i.e., delay) activation, while allowing for rapid activation in response to an external signal. While the glycerol-salt condition stabilizes the inhibitory complex for several weeks, SDS addition always expedites activation (Fig. 2c). Furthermore, the stochastic behavior is enhanced under stabilizing conditions. While glycerol induces structure into the isolated LMC-domain, it alters neither subtilisin activity, nor the secondary structure of the inhib ition complex and mature subtilisin (Fig. 3d). This indicates that glycerol shifts the equ: ilibrium towards the formation of the inhibition complex by inducing structure in the isolated LMC-domain. The X-ray-structure of the inhibition complex establishes that the B-factor of the LMC in complex is twice that of subtilisin and implies substantially larger backbone dynamics within the LMC-domain. In Example 5, glycerol was then examined to see if it reduces intrinsic conformational dynamics of the LMC-domain in the inhibition complex, to decrease proteolytic susceptibility. Fig. 3e shows that trans-proteolysis of the LMC within the inhibition complex by TPCK-treated trypsin is substantially reduced in glycerol and is consistent with decreased conformational entropy.

Energetics of stochastic activation. To investigate causes for the observed stochastic behavior, activation energies (Ea) for precursor maturation were established under Example 6 herein below. The Arrhenius plot shown in Fig. 4a is non-linear with the transition temperature of 25°C (298 °K). The activation energies computed from the two slopes are 19.2 ± 0.3 kcal mol^"1 and 11.6 ± 0.1 kcal mol^"1. The change in slope indicates that the dependence of protease activation changes from one RDS to another, and may arise from instability of the LMC-domain in the inhibition complex at higher temperatures.

To complete the free-energy diagram for precursor activation, the extent to which LMC-binding stabilizes the inhibition complex was established. Unlike molecular chaperones, LMCs can bind tightly with their cognate proteases and the affinity is similar to the best known protease inhibitors. The inhibition constant for subtilisin E with its cognate LMC was estimated using slow-binding inhibition kinetics as shown in Fig. 4b. The rate coefficient K, for this interaction under folding conditions was 8.36 x 10^"9M, and is close to that observed for the subtilisin E-streptomyces subtilisin inhibitor interaction (K_t of 4.2 x 10^"9 M). This value is also consistent with affinity coefficient estimated for LMC: subtilisin BPN' interaction using micro-calorimetry. By using the thermodynamic relation ΔG = -RT hi ( K_eq), w here K _eq= K ι , i t w as e stimated t hat I MC b inding w ith s ubtilisin s tabilizes t he inhibitor complex by ~11 kcal mol^"1 as shown in Fig. 4b. The computed free-energy is in close agreement with k_on and k_off rates determined using fluorescence spectroscopy f or a subtilisin homolog. The free-energy diagram for precursor maturation favors formation of a thermodynamically stable inhibition complex (see Fig. 4c) over the active protease.

Protease activation is not a spontaneous process because the release of the inhibitory LMC-domain from the complex is energetically unfavorable. The stochastic behavior arises because the RDS represents the probabilistic release/degradation of the LMC-domain from the thermodynamically stable inhibition complex. The formation of a free subtilisin molecule forces the equilibrium towards release because the LMC-domain in the inhibition complex becomes an excellent substrate for the free protease. Stochastic events are known to regulate highly predictable patterns of gene expression, signal transduction, cellular replication and differentiation. Therefore, deterministic patterns in biological systems are achieved through events that have probabilistic features. In the case of protease activation, stochastic behavior may be tightly coupled with diverse environmental signals to regulate protease activation precision through selective modulation of conformational entropy.

Standard liquid detergent according to aspects of the present invention. An

Example of a standard liquid detergent can be formulated as follows: AE, Berol 160; LAS, Nasa 1169/P; coconut fatty acid; Oleic acid; triethanolamine; glycerol (e.g., 10%); ethanol; tri.Na.Citrat.2H₂O; CaC1.2H₂ O; NaOH; water from LAS; water from glycerol; water added; and prosubtilsin. The glycerol in the formula would, according to the present invention, induce structure in the LMC-domain thereby prolonging (i.e., delaying) release of active protease. The inventive formulation would also contain granules containing SDS (an external activation signal). In use, the SDS sequestered in the granules would be activated by at least one of an action such as mixing the formulation to rupture the granules, raising the temperature to rupture the granules, and/or rubbing the formulation to rupture the granules, among others. Once the SDS contained in the granules is released, the interaction of the SDS and the pro-subtilisin would promote activation of the subtilisin.

Stabilizer and activator signals. Various 'stabilizer' and external "activation" signals are discussed in the following Examples with respect to stabilization and subsequent activation of stabilized proteases (e.g., activation of stabilized pro-subtilisin, or ProD- subtilisin, etc.).

Stabilizers. According to the present invention, p referred stabilizers are glycerol, where the glycerol concentration is preferably from about 5% to about 25%. Preferably, the glycerol concentration is from about 8% to about 20%, or from about 10% to about 15%, and most preferably, about 10%. Generally, increasing glycerol increases the time required for subsequent activation.

Similarly, (NH )₂SO₄ functions as an inventive stabilizer, and is preferably present at a concentration of about 0.5 to about 1.5 M. Preferably, (NH₄)₂SO₄ is present at about 0.75 to about 1.2M, and most preferably at about 1.0M.

Combinations of glycerol and (NH )₂SO are also disclosed as inventive stabilizers, and preferably reflect, for each stabilizer, the above-identified preferred ranges.

Activators. Likewise, various 'activators' or 'activation signals' are discussed in the following Examples with respect to subsequent activation of stabilized proteases (e.g., subtilisin).

According to the present invention, preferred activators include, alone or in combination, sequestered (e.g., granules) SDS, salt-shock (i.e., reduce the (NH₄)₂SO₄ concentration), active subtilisin, and/or pH-shock-Tris-HCL.

SDS is a preferred activator and is preferably present at concentration of from about 0.005% to about 0.05%. Preferably, SDS is at about 0.01% to about 0.025%, and preferably at about 0.01%. Concentration of SDS higher that about 0.05% were found to destabilize the protein.

For Salt Shock, the (NH₄)₂SO₄ concentration is preferably reduced to below about 0.25 M Higher (NH₄)₂SO₄ concentrations slow protease activation. Salt shock is achieved by dilution of the enzyme into a relatively low-salt condition.

Active subtilisin concentration is preferably at about 1 to 10 nM. Preferably, active subtilisin concentration is at about 2 to about 5 nM, and most preferably at about 2.5 nM. Concentrations of active subtilisin of about 10 nM also work for the present purposes. The target pH for 'pH-shock' is from about pH 7.5 to about pH 10.0. Preferably, the target pH is about pH 8 to about pH 9, and preferably, at about pH 8.5.

EXAMPLE 1 (Rapid dilution folding technique) Proteins were expressed and purified as described in Shinde, U. P. et al., Nature 389:

520-522, 1997, incorporated in its entirety herein by reference. A volume of 20 μl of denatured prosubtilsin (100 μM) was rapidly mixed in 19,980 μl of the refolding buffer (50 mM MES-NaOH, pH 6.5, 0.5 M (NH₄)₂S0₄, 1 mM CaCl₂) at 23°C with 0.5 mM synthetic substrate (N-succ-AAPF-pNA). After 15 minutes, the stirring was stopped and 200 μl aliquots of the reaction mixture were transferred to a 96-well microplate, after 30 minutes of folding initiation. Subtilisin activity was monitored by release of p-nitroanilide measured at 405 nm in a microplate reader maintained at the desired temperature. The time of activation was calculated from the X-axis intercept of the 'none to all' transition as described in Inouye, M et al., Nat. Struct. Biol. 8:321-325, 2001.

EXAMPLE 2 (Amount of active subtilisin) Precursors were folded at 23°C as described above in Example 1, however without the synthetic substrate. The reaction was allowed to proceed on the microplate at the desired temperature. After 24 hours of incubation, synthetic substrate was added to a final concentration of 0.5 mM and the velocity of substrate hydrolysis in each well was measured. Care was taken to ensure that substrate concentration was not rate-limiting during velocity measurements. Under these conditions, velocity of substrate cleavage was proportional to enzyme concentration and 80 nM of mature subtilisin E gave approximately 20 mOD min^"1.

EXAMPLE 3 (Signal induced modulation of precursor activation) Precursors (200 nM) were folded and aliquoted (100 μl each well) onto the microplate. Signals (2 x concentration) were prepared in the folding buffer and 100 μl were added to each well at the desired time and mixed. The final concentration of the signals that expedited activation were SDS-0.01%; salt-shock-0.25M (NH )₂SO₄; active subtilisin-2.5 nM, pH-shock-Tris-HCI (50 mM, pH 8.5). While lμl of SDS (1%) in each well was sufficient to activate the precursor, a lOOμl volume was chosen as the signal to enable complete mixing. 100 μl of glycerol (20%) (e.g., final 10%), (NH₄)₂SO₄ (2.0 M) (e.g., final 1M) and the combination of glycerol-(NH₄)₂SO₄ serve as signals that prolong activation. Stabilizers were added 30 minutes after folding initiation to allow complete folding and autoprocessing and deterministic activation was induced using SDS (e.g., 0.01%).

EXAMPLE 4 (Circular dichroism measurements) CD measurements were performed on an automated AVIV 215 spectrophotometer maintained at 25°C and spectra were taken, between 190 to 260 nm. Protein concentrations were maintained between 0.25 to 0.4 mg ml^"1. A 1 mm path-length cuvette was used to measure spectra except in case of the LMC-domain, where a 0.5 mm path-length cuvette was used. The spectra depicted in Fig. 3d represent averages of 3 independent scans.

EXAMPLE 5 (Proteolytic stability of the precursor)

A cleaved but proteolytically inactive complex (~1 μM) was prepared as described in Hu Z, et al., F. J Biol Chem. 271: 3375-3384, 1996. The sample was maintained under normal and stabilized conditions (e.g., 10% glycerol) and 1/10 the amount of TPCK-treated trypsin was added. A volume of 50 μl of the complex were removed at fixed time intervals and the reaction was stopped by TCA precipitation. (Yabuta, Y., et al., J. Biol. Chem. 276:44427-44433, 2001). The extent of LMC-degradation was quantified using densitometry.

EXAMPLE 6 (Activation Energy)

Ea was calculated from the Arrhenius plots obtained by measuring the rate, k, of the propeptide/active substilisin conversion at different temperatures from the slope (Ea/R) of the plot of log (k) versus 1/T (Figure 4a). Example 7 (Inhibition Constants) Mature subtilisin was rapidly added (up to 260 nM) to the protease assay buffer that contains 2.5 to 5 μM of the free propeptide along with 2 mM synthetic substrate. The release of p-nitroanilide was monitored as a function of time as described earlier. The initial (No) and steady state (Ns) velocities were obtained by fitting the data to an equation for slow binding inhibition. K_\ was estimated from the plot of (NoNs) versus inhibitor concentration.

EXAMPLE 8

(Νon-limiting Examples; illustration the Detergent Compositions including the stabilized Enzyme) Detergent Dl:

A detergent powder is formulated to contain: anionic detergent, nonionic detergent, phosphate-containing builder, acrylic or equivalent polymer, perborate bleach precursor, amino-containing bleach activator, silicate or other structurant, pro-subtilisin of about 8 glycine units/mg activity, with alkali to adjust to desired pH in use, and (ΝH₄) SO₄ (signal prolonging activation) (e.g., 1 M). The formulation also contains granules containing sequestered salt-shock. The enzymes include wild and mutant subtilisin protease, among others, but are not limited thereto. Preferably, the sequestered salt-shock is engineered to bring the (NH4)₂SO concentration to 0.25 M. Preferably, the subtilisin is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D la:

A detergent powder is formulated to contain: anionic detergent, nonionic detergent, phosphate-containing builder, acrylic or equivalent polymer, perborate bleach precursor, amino-containing bleach activator, silicate or other structurant, protease enzyme of about 8 glycine units/mg grade, with alkali to adjust to desired pH in use, and glycerol (signal prolonging activation) (e.g., 10%). The formulation further contains granules sequestering SDS therein.

The enzymes include wild and mutant subtilisin protease, among others, but are not limited thereto. Preferably, the granules are engineered to rapidly release SDS to a final concentration of about 0.01%. Preferably, the protease is a subtilisin. Preferably, the subtilisin is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD- loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D2:

A detergent powder is formulated to contain: anionic detergent, nonionic detergent, zeolite-containing builder, acrylic or equivalent polymer, perborate bleach precursor, amino-containing bleach activator, silicate or other structurant, protease enzyme, with alkali to adjust to desired pH in use, and glycerol-(NH₄)₂SO₄ (signal prolonging activation) (e.g., 1.0 M). The formulation further contains granules sequestering active subtilisin therein.

The enzymes include wild and mutant stable subtilisin protease, among others, but are not limited thereto. Preferably the granules are engineered to release subtilisin to a concentration of about 2.5 nM. Preferably, the protease enzyme (but not the sequestered subtilisin) is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD- loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D2a:

A detergent powder is formulated to contain: anionic detergent, nonionic detergent, zeolite-containing builder, acrylic or equivalent polymer, perborate or peracid bleach precursor, amino-containing bleach activator, silicate or other structurant, protease enzyme, with alkali to adjust to desired pH in use, and a combination of glycerol and (NH₄)₂SO₄ (signal prolonging activation) (e.g., 10% and 1.0 M, respectively). The formulation further contains granules sequestering pH-shock-Tris-HCL therein. The enzymes include wild and mutant stable subtilisin protease, among others, but are not limited thereto. Preferably, the sequestered pH-shock is engineered to produce Tris- HCI at 50 mM, pH 8.5). Preferably, the subtilisin is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D3:

An aqueous detergent liquid is formulated to contain: Dodecylbenzene-sulphonic acid, C12-C15 linear alcohol condensed with 7 mol/mol ethylene oxide, monoethanolamine, citric acid, sodium xylenesulphonate, sodium hydroxide, pro-subtilisin, glycerol (e.g., 10%), protease, and water. The formulation further includes pH-shock-Tris-HCL sequestered from the remainder of the formulation.

In use, the liquid detergent formulation containing the protease is allowed to react with the sequestered Tris-HCL. The enzymes include wild and mutant stable subtilisin protease, among others, but are not limited thereto. Preferably, the sequestered pH-shock is engineered to produce Tris-HCI at 50 mM, pH 8.5). Preferably, the subtilisin is ProD- subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D4:

A nonaqueous detergent liquid is formulated using C13-C15 linear primary alcohol alkoxylated with ethylene oxide, propylene oxide, triacetin, sodium triphosphate, soda ash, sodium perborate monohydrate containing a minor proportion of oxoborate, TAED, EDTA of which 0.1% as phosphonic acid, Aerosil, SCMC it, protease, and (NH₄) SO₄ (e.g., 1.0 M) The formulation further includes sequestered salt-shock. m use, the liquid detergent formulation containing the protease is allowed to react with the sequestered salt-shock (i.e., solution is diluted). The enzymes include wild and mutant stable subtilisin protease, among others, but are not limited thereto. Preferably, the sequestered salt-shock is engineered to bring the (NH4)₂SO₄ concentration to about 0.25 M or below. Preferably, the protease is subtilisin. Preferably, the pro-subtilisin is ProD- subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D5: A detergent powder is formulated in the form of a granulate containing a surfactant of sodium dodecylbenzene sulphonate and a mixture of Synperonic A7 and Synperonic A3 and zero neutral inorganic salt (e.g., sodium sulphate), plus phosphate builder, sodium perborate tetrahydrate, TAED activator, sodium silicate and minors including sodium carbonate and moisture. Enzymes (e.g., proteases) are included along with glycerol (e.g., 10%). The formulation further includes sequestered SDS (e.g., granules).

In use, the SDS is allowed to intermix with the detergent powder formulation containing the pro-subtilisin with glycerol. The enzymes include wild and mutant stable pro-subtilisin protease, among others, but are not limited thereto. Preferably, the granules are engineered to rapidly release SDS to a final concentration of about 0.01%. Preferably, the protease is a subtilisin. Preferably, the subtilisin is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D6:

A detergent (soap) bar according to an embodiment of the invention is formulated as follows: soap including pan-saponified tallow, coconut oil, neutralized with orthophosphoric acid, mixed with protease (e.g., pro-subtilisin) and with sodium formate, borax, propylene glycol and sodium sulphate, is then plodded on a soap p roduction line. The soap will also contain sequestered subtilisin (e.g., granules).

The enzymes include wild and mutant stable subtilisin protease, among others, but are not limited thereto. In use, the sequestered subtilisin will interact with the pro-subtilisin causing a cascading reaction activating the prosubtilisin to its active state. Preferably the granules are engineered to release subtilisin to a concentration of about 2.5 nM. Preferably, the protease enzyme (but not the sequestered subtilisin) is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

Detergent D7: An isotropic aqueous liquid detergent suitable for laundry can be formulated as follows: citric acid; boric acid; NaOH; KOH; glycerol (e.g., 10%); ethanol; nonionic surfactant; C12-alcohol 6.5 EO; ethoxylate groups/mol) or sodium primary alcohol sulphate; oleic acid; Coconut oil (C12) soap; protease; minors and water.

The formulation further can include, alone or in combination, sequestered (e.g., granules) SDS, salt-shock ((NH₄)₂SO₄, active subtilisin, and/or pH-shock-Tris-HCL. The sequestration can be accomplished by any manner known to those of skill in the art, including but not limited to art-recognized phase-separation, and other delivery systems. The delivery systems can include but are not limited to polymeric matrix systems, wax matrix systems, multi-particulate systems, and combinations thereof. The most commonly used delivery systems can be broadly classified as diffusion, reservoir, pore-forming wax, or coated-bead systems.

Diffusion devices are composed of a drug dispensed in a polymer which diffuses from the entire physical tablet. Reservoir devices usually consist of a semi-permeable barrier which is involved in the release of the active from a core site within the tablet. Coated-bead systems employ an enteric or pH-sensitive coating of aggregated particles of the active ingredient packaged in capsule form. Pore-forming wax systems incorporate the active ingredient into a wax base and rely upon the rate of diffusion to control the release of the "active ingredient." The pH can be adjusted to a value between 9 and 10. The enzymes include wild and mutant stable subtilisin protease, among others, but are not limited thereto. Preferably, the granules are engineered to rapidly release SDS to a final concentration of about 0.01%. Preferably, the sequestered salt-shock is engineered to bring the (NH4)₂SO₄ concentration to 0.25 M. Preferably, the sequestered pH-shock is engineered to produce Tris-HCI at 50 mM, pH 8.5). Preferably the granules are engineered to release subtilisin to a concentration of about 2.5 n M. P referably, t he p rotease e nzyme ( but n ot t he s equestered s ubtilisin) i s ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

EXAMPLE 9

(Contact Lens Cleaner) Current formulations of contact lens cleaners containing an active protease, like subtilisin, do not have a long shelf life. Use of an inactive protease, such as pro-subtilisin, while having an activating substance sequestered in, for example, a granule, allow for increased shelf life of the protease in a contact lens cleaner. When in use, the formulation could be rubbed in the users hand or between the fingers to break the granules containing the activating substance or signal. The signal then would come in contact with the prosubtilisin in the formulation. This would then cause activation of the pro-subtilisin. Besides sequesting the activating substance or signal, temperature changes and/or water changes, could cause the signal and the activated subtilisin to come into contact and activate the pro-subtilisin into its active state. Activation signals, as discussed above, include, alone or in combination, sequestered (e.g., granules) SDS, salt-shock (NH₄)₂SO , active subtilisin, and/or pH-shock-Tris-HCL. Preferably, the protease enzyme (but not the sequestered subtilisin) is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD- loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

We have found that we can activate previously inactive proteases. One way in which this is accomplished is through cleavage of the LMC from the pro-subtilisin, the rate determining step. This causes the release of active subtilisin. The first released subtilisin can t hen "go b ack" and a ct o n t he o ther p ro-subtilisin m olecules to e ause t he r elease o f more active subtilisin.

In personal care products, the pro-subtilisin and the LMC can be kept separate. Then, when it is desired to active the pro-subtilisin in the product, the LMC and the pro- subtilisin can be caused to come into contact and reach by, for example, but not limited to, breaking granules, increasing the temperature or increasing the water content. For example, many personal care products can be made with the pro-subtilisin in non aqueous solution. To activate the pro-subtilisin, granules containing water and LMC would contact the non- aqueous solution containing the subtilisin so as to begin the cascade reaction to active subtilisin.

EXAMPLE 10

(Denture Cleaner)

In another example, denture cleaning compositions for cleaning dentures outside of the o ral c avity i nclude one o r m ore s tabilized p rotease enzymes. S uch d enture cleaning compositions include an effective amount of one or more protease enzymes, such as prosubtilisin, and a denture cleansing carrier. Various denture cleansing composition formats such as effervescent tablets and the like are well known in the art (e.g., U.S. Patent No. 5,055,305, Young, incorporated herein by reference), and are generally appropriate for incorporation of one or more protease enzymes for removing proteinaceous stains from dentures. The pro-subtilisin is in a stabilized form under the influence of prolongation signals described in Example 3. The pro-subtilisin can be activated through contact with one of the activating signals of Example 3. Activation signals, as discussed above, inplude, alone or in combination, sequestered (e.g., granules) SDS, salt-shock (NH₄)₂SO₄, active subtilisin, and/or pH-shock-Tris-HCL. Preferably, the protease enzyme (but not the sequestered subtilisin) is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

EXAMPLE 11 (Skin Care Product, Light Cream)

A skin care product, such as a sunscreen emulsion, can be made from the following ingredients, which are indicated by their chemical or Cosmetic, Toiletry and Fragrance Association (CTFA) name: [Water phase]; methylparaben (preservative); pantethine (moisturizer); carbomer 934 (thickener); sodium hydroxide (neutralizer); pro-subtilisin (protease); [Oil phase]; heavy mineral oil; stearic acid (anionic emulsifier); cholesterol (auxiliary emulsifier); cetyl alcohol (auxiliary emulsifier); castor oil (emollient); cetyl palmitate (emollient); octyl dimethyl PAPA (U.V.-absorber); and propylparaben (preservative). hi a mixing vessel equipped with a mechanical stirrer, water and the water phase ingredients other than the sodium hydroxide and protease (e.g., pro-subtilisin) are added and mixed with heating to about 75°C to form a uniform aqueous dispersion. The sodium hydroxide solution is then added and mixed into the aqueous phase to neutralize the acidic Carbomer thickener. In a separate mixing vessel, the mineral oil and oil phase ingredients are added and mixed with heating to about 80°C to form a uniform oil phase. The heated oil phase is slowly added to the heated water phase using high speed mechanical dispersing means. Mixing is continued until a homogeneous oil/water emulsion is obtained. The emulsion is cooled to room temperature. If desired, optional colorants such as water-soluble dyes are preferably mixed into the emulsion at about 45-50°C and fragrant oils are preferably added at about 35-40°C with an inactivating signal described in Example 3. Granules containing and sequestering an activating signal described in Example 3 are included in the skin care product. When applied to the skin, the rubbing action and the heat generated from rubbing and contact with the skin cause rupture of the granules and dispersal of the activating signal. Upon contact with the pro-subtilisin, the pro-subtilisin undergoes cleavage of the LMC and is activated to the active subtilisin state. Activation signals, as discussed above, include, alone or in combination, sequestered (e.g., granules) SDS, salt-shock (NH₄)₂SO₄, active subtilisin, and/or pH-shock-Tris-HCL. Preferably, the protease enzyme (but not the sequestered subtilisin) is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD-loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

EXAMPLE 12

(Wound Cleaning)

Subtilisin can be used in wound cleaning compounds to aid in the reduction and decomposition of necrotic tissue as discussed in, for example, U.S. Patent No. 4,904,469 incorporated herein in its entirety. A suitable wound cleaning formulation may contain polymyxin B sulfate, bacitracin zinc, neomycin, and pro-subtilisin in a base of cocoa butter, cottonseed oil, olive oil, sodium pyruvate, tocopheryl acetate, and white petrolatum.

Sequestered from the wound cleaning formulation in any delivery method known to one of skill in the art is contained one or a combination of SDS, salt-shock (NH₄)₂SO₄, active subtilisin, and/or ph-shock-Tris-HCL.

When it is desired to activate the pro-subtilisin to active subtilisin in the wound cleaning formulation, the sequestered products and the pro-subtilisin interact to cause active subtilisin to be formed. Activation signals, as discussed above, include, alone or in combination, sequestered (e.g., granules) SDS, salt-shock (NH₄) SO₄, active subtilisin, and/or pH-shock-Tris-HCL. Preferably, the protease enzyme (but not the sequestered subtilisin) is ProD-subtilisin (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003), or ProD- loaded subtilisin (Id), or a variant thereof (see Example 14 herein below).

EXAMPLE 13 (Dishwashing Composition) A dishwashing composition can be formulated including from about 0.0001% to about 10% protease enzyme, for example pro-subtilisin in a stabilized form through the action of signal as described in Example 3; from about 0.1% to about 10% surfactant; and optionally, one or more cleaning composition materials compatible with the protease enzyme selected from the group consisting of solvents, buffers, enzymes, dispersing agents, suds suppressors, enzyme stabilizers, bleaching agents, dyes and perfumes. The prosubtilisin is then activated through contact with one of the activating signals described in Example 3.

EXAMPLE 14 (Activation of a stabilized protease comprising a computer designed peptide chaperone) A decision-based computer algorithm that maintained conserved residues, but varied all non-conserved residues from a multiple protein sequence alignment of 176 homologues of subtilisin was previously developed and utilized to design a novel 77-residue peptide sequence (ProD) homologous to the subtilisin LMC domain (Yabuta et. al., J Biol. Chem. 278:15246-51, 2003; incorporated herein by reference in its entirety). Interestingly, despite a difference of 5 pH unit between their isoelectric points and displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor.

According to preferred embodiments of the present invention, despite the fact that ProD is a 10-fold better inhibitor of subtilisin than ProWT (Id), stabilized ProD-subtilisin (or ProD-loaded subtilisin) was found to be rapidly activated by an external activation signal (0.01% SDS). According to preferred embodiments, ProD-subtilisins function in place of, or in combination with Pro-subtilisins in the various external activation signal protocol embodiments and activatable composition embodiments described herein above.

Alternatively, mature subtilisins are loaded with ProD polypeptide, and used in the various external activation signal protocol embodiments and composition embodiments described herein above.

The peptide sequence (ProD) was designed through a decision based computer algorithm that maintained conserved residues but varied all non-conserved residues from a multiple protein sequence alignment (Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003). According to the present invention, the DNA sequence coding for ProD is synthesized and subsequently expressed in Escherichia coli and the resulting peptide purified as previously described (Id). Despite a difference of 5 pH units between their isoelectric points (pi) and displaying only 16% sequence identity with the wild-type propeptide (ProWT), ProD chaperones folding and functions as a potent subtilisin inhibitor (Id). The computed 2D structure and the distribution of hydrophobic residues within these two propeptides are similar. However, unlike ProWT, ProD adopts a well-defined β-conformation as an isolated peptide (Id). The CD structure of ProD: subtilisin is similar to ProWT: subtilisin (Id). Hence despite low sequence identity and opposite net charge, ProD and ProWT adopt similar structural scaffolds (Id). The amino acid sequence of the 77-residue ProD polypeptide is provided herein as

SEQ ID NO:l (see also Figure 5 where it is aligned with the corresponding wild type sequence, SEQ LD NO:2). According to the present invention, ProD chaperones the folding of denatured subtilisin, and inhibits subtilisin activity. According to the present invention, ProD inhibits subtilisin activity under the above-described conditions of glycerol (10%), (NH₄)₂SO₄ (1.0 M) and the combination of glycerol-(NH₄)₂SO₄, that serve as stabilizing signals that prolong (i.e, delay) the activation process.

According to the present invention, ProD-subtilisins and ProD-loaded subtilisins undergo expedited activation by the above-described signals that expedite activation of ProWT-subtilisins; namely, SDS-0.01%; salt-shock-0.25M (NH₄)₂SO₄; active subtilisin-2.5 nM; and pH-shock-Tris-HCI (50 mM, pH 8.5).

ProD variants, having from one to about 5, or from one to about 10, or from one to about 15, or from one to about 20 conservative substitutions at variable (see Fig. 5, showing conserved residues) residues, which, like ProD, show some degree of enhanced inhibition of subtilisin relative to ProWT-comprising polypeptides are also encompassed by the present invention. Invariant regions of the ProD sequence are indicated under features of SEQ LD NO:l in the Sequence Listing (see also Figure 5; and see Yabuta et. al., J. Biol. Chem. 278:15246-51, 2003). Conservative amino acid substitutions are well known in the relevant art, and will be obvious to those of ordinary skill in the relevant art.

Illustrative of a preferred embodiment, ProD-subtilisin, stabilized in 10% glycerol, was shown to be rapidly activated by 0.01% SDS.

The discussions and Examples above are descriptive, illustrative and exemplary and are not to be taken as limiting the scope defined by any appended claims.

Claims

CLAIMS We claim:

1. An activatable protease-containing composition, comprising: at least one of a pro-subtilisin protease comprising an N-terminal propeptide that is an intramolecular chaperone (LMC), and a subtilisin protease that is non-covalently loaded with an N-terminal propeptide suitable as an intramolecular chaperone (LMC), wherein the proteases are inactive, or substantially so; a stabilizing agent; and a sequestered activator sufficient to rapidly activate the inactive protease upon triggered release of the activator.

2. The composition of claim 1, wherein the stabilising agent is selected from the group consisting of from about 5% to about 25% glycerol, (NIL;)₂SO₄ from about 0.5 to about 1.5 M, and combinations thereof.

3. The composition of any one of claims 1 or 2, wherein the activator, in the non-sequestered state, is selected from the group consisting of SDS at about 0.005% to about 0.05%, active subtilisin from about 1 to about 10 nM, an agent that establishes a pH value between about 7.5 and about 10.00, and dilution, in the case of an (NH₄)₂SO₄ stabilizer, of the (NH₄) SO₄ concentration to a value at or below about 0.4 M.

4. The composition of any one of claims 1-3, wherein the stabilizing agent is selected from the group consisting of glycerol at about 10%, (NH₄) SO at about 1.0 M, and combinations thereof.

5. The composition of any one of claims 1-4, wherein the activator, in the non- sequestered state, is selected from the group consisting of SDS at about 0.01%, active subtilisin at about 2.5 nM, an agent that establishes a pH value at about 8.5, and dilution, in the case of an (NH₄)₂SO₄ stabilizer, of the (NH₄)₂SO₄ concentration to a value at or below about 0.25 M.

6. The composition of any one of claims 1-5, wherein sequestration is by a system selected from the group consisting of sequestering granules, sequestering polymers, diffusion systems, reservoir devices, porous wax systems, coated-bead systems, and combinations thereof.

7. The composition of any one of claims 1-6, wherein the pro-subtilisin is ProD-subtilisin corresponding to SEQ LD NO:l, or is a variant thereof having from one to about 20 conservative amino acid substitutions at non-conserved positions within SEQ LD NO:l.

8. The composition of any one of claims 1-6, wherein the subtilisin is loaded with ProD polypeptide according to SEQ LD NO:l, or with a variant thereof having from one to about 20 conservative amino acid substitutions at non-conserved positions within SEQ ID NO: 1.

9. A detergent formulation, comprising a cleaning effective amount of a composition of any one of claims 1-8.

10. A dishwashing formulation, comprising a cleaning effective amount of a composition of any one of claims 1-8.

11. A contact lens cleaning formulation, comprising a cleaning effective amount of a composition of any one of claims 1 -8.

12. An oral care formulation, comprising a cleaning effective amount of a composition of any one of claims 1-8.

13. A skin care or cosmetic formulation, comprising a cleaning effective amount of a composition of any one of claims 1-8.

14. A method of activating, upon demand, an inactive subtilisin protease, comprising: obtaining a composition having at least one of a pro-subtilisin protease comprising an N-terminal propeptide that is an intramolecular chaperone (LMC), and a subtilisin protease that is non-covalently loaded with an N-terminal propeptide suitable as an intramolecular chaperone (LMC), wherein the proteases are inactive, or substantially so, and wherein the composition also comprises a stabilizing agent, and a sequestered activator sufficient to rapidly activate the inactive protease upon triggered release of the activator; and triggering the activator.

15. A method of manufacturing activatable protease-containing compositions having a prolonged protease shelf-life, comprising: obtaining a composition comprising at least one of a pro-subtilisin protease comprising an N-terminal propeptide that is an intramolecular chaperone (LMC), and a subtilisin protease that is non-covalently loaded with an N-terminal propeptide suitable as an intramolecular chaperone (LMC), wherein the proteases are inactive, or substantially so; adding a stabilizing agent thereto; and adding thereto a sequestered activator sufficient to rapidly activate the inactive protease upon triggered release of the activator, whereby the protease shelf-life is prolonged in a state that is activatable upon demand by triggered release of the sequestered activator.