MXPA97004876A - Stabilization of enzi - Google Patents

Abstract

The present invention relates to methods for the stabilization of enzymes in liquid compositions, including those liquid compositions having a high water content and those liquid compositions of stabilized enzymes formed by means of which. The method involves the formation of the enzyme, so that it is in an insoluble form thereof and then the addition thereto of an agent to maintain the enzyme in the insoluble form thereof. Examples of such insoluble forms are crystal forms of the enzyme. Enzymes that are stabilized in this manner are useful for combination with liquid compositions, including liquid compositions having a high water content. The method is particularly useful for the preparation of liquid detergent compositions containing enzymes,

Description

STABILIZATION OF ENZYMES Field of the Invention The present invention relates to the stabilization of enzymes and, in particular, to methods for increasing the stability of enzymes in liquid compositions, such as detergent formulations, and to liquid compositions containing stabilized enzymes, including liquid detergent formulations containing enzymes, which are prepared by the present.

Background of the Invention A primary or principal commonly found in the production of liquid compositions containing enzymes, such as detergent formulations, is the formation of aerosols (or nebulizations) containing enzymes. Such aerosols containing enzymes are a serious matter for the safety of the work area. This problem is especially acute where the processing and / or formulation conditions are REF: 25070 drive on a commercial scale and commercial producers have required special precautions to reduce the risks for which they are proposed. Another primary problem encountered in the production and use of liquid compositions containing enzymes is the maintenance of the stability (and therefore the activity) of the enzyme within the liquid composition. The stability problems of the enzyme are especially acute where the enzymes are included in liquid compositions having a high water content and / or high pH. The water content in such compositions stabilizes the enzyme, resulting in a loss of enzyme activity. The high pH of such compositions leads to the denaturation of the enzyme, resulting in a loss of enzyme activity. The formulations of liquid detergents present particular problems with regard to the stabilization of enzymes. This is because such detergent formulations frequently have both a high water content and a high pH. These conditions not only solubilize the enzyme but also result in the denaturation of the enzyme as well as adversely affect stability and enzymatic activity. In view of the foregoing, considerable efforts have been made to improve the stability of the enzymes in liquid compositions, and in particular detergent formulations, to preserve the enzymatic activity thereof during transportation and storage. Examples of compositions for improving stability (shelf life) of the enzymes in liquid slurry detergent formulations include the use of various types of additives. Such proposed additives include alkali metal pentaborate (U.S. Patent No. 4,404,114). Combinations of calcium ions and acids or carboxylic salts of low molecular weight (U.S. Patent Nos. 4,318,818 and 4,305,837) and combinations of an antioxidant and a hydrophilic polyol (U.S. Patent No. 4,243,543). Finally, the use of reducing alkali metal salts, such as sodium sulfite, has been proposed in particular cases where boric acid or formulations containing alkali metal borate are involved (US Patent No. 4,462,922). Unfortunately, the use of such stabilizers and additives is not always desirable or useful in liquid compositions. This is due, at least in part, to the fact that these can lead to the formation of contaminated effluents and / or present other problems of use. In addition, the use of such stabilizers and additives can increase the cost of the compositions that are prepared with which. Finally, the use and presence of stabilizers in such compositions containing enzymes, as proposed in the references discussed above, does not reduce or eliminate the problem of the formation of aerosols containing enzymes (nebulizations). No description is known of the use of enzyme crystals, either by themselves or in combination with stabilizers, has been taught, described or suggested for the improvement of the stability of enzymes in liquid compositions. In fact, and perhaps due to the fact that when enzyme crystals are rapidly solubilized in such compositions (resulting in poor storage stability), the use of enzyme crystals in aqueous formulations has been rejected. While it has been described to use enzyme crystals to obtain improved enzyme stability in formulations of dry (granulated) and anhydrous (thick solutions) detergents (patent application No. WO 91/09941) their use in liquid compositions and in particular in compositions Liquids having a water content (aqueous compositions) has been avoided. This is due, at least in part, to the fact that such enzyme crystals (similar to the conventional enzyme forms discussed in detail above) are easily solubilized. In themselves, they exhibit poor storage stability when used in liquid compositions, and in particular liquid compositions having a high water content. Accordingly, it can be seen that a need remains to provide a solution to the problem of reducing and / or eliminating the formation of aerosols (nebulizations) containing enzymes when producing liquid compositions containing enzymes, and especially liquid detergent formulations containing enzymes In this regard, it can be seen that a need remains to provide a method of improving stable, liquid compositions containing enzymes, including formulations of liquid detergents, while reducing or eliminating the formation of aerosols (nebulizations) containing enzymes that are commonly associate with them. Further in this regard, it can also be seen that a need remains to provide stable, liquid compositions containing enzymes, including liquid detergent formulations, which have reduced levels of aerosols containing enzymes. Furthermore it can be seen that a need also remains to provide a solution to the problem of improving the stabilization of enzymes in liquid compositions, and in particular liquid detergent formulations, to preserve the enzymatic activity thereof. In this regard, it can be seen that a need remains to provide a method for providing stable liquid compositions containing enzymes, including formulations of liquid detergents. Further in this regard, it can also be seen that a need remains to provide stable liquid enzyme containing compositions, including liquid detergent formulations, wherein the enzyme retains a substantial portion of its enzymatic activity for long periods.

Brief Description of the Invention It is a primary or main objective of the present invention to provide stable liquid compositions containing enzymes and in particular, liquid compositions containing a water content (aqueous compositions). Another primary objective of the invention is to provide liquid detergent compositions containing stable enzymes, and in particular, liquid detergent formulations having a water content (aqueous compositions). It is a further, primary objective of the present invention to provide a method for providing stable liquid compositions containing enzymes and in particular, liquid compositions having a water content (aqueous compositions).

Another primary objective of the present invention is to provide a method for providing liquid detergent formulations containing enzymes, stable and in particular, liquid detergent compositions having a water content (aqueous compositions). In another aspect of the present invention, it is a further objective of the present invention to reduce or eliminate the problems of the formation of aerosols (nebulizations) containing enzymes during the production of liquid compositions containing enzymes, including formulations of liquid detergents containing enzymes In accordance with the teachings of the present invention, a method for the production of stable, enzyme-containing liquid compositions is described herein. This method includes the formation of soluble forms of the enzyme of interest that are incorporated into the liquid composition. This method further includes combining, in a liquid formulation, the enzyme in the insoluble form thereof with an agent to maintain the enzyme in its soluble form. In this manner, stable, liquid, enzyme compositions of the present invention are provided.

Preferably, insoluble forms of the enzyme can be formed by crystallization of the enzyme, whereby enzyme crystals are formed and provided. Alternatively, insoluble forms can be provided by forming amorphous forms of the enzyme, whereby an amorphous enzyme is formed and provided. In a preferred embodiment, the method comprises forming the insoluble forms of the enzyme of interest while in the presence of an agent to maintain the enzyme in its insoluble form. In another preferred embodiment, the method comprises forming the insoluble forms of the enzyme of interest before combining the insoluble form of the enzyme with the agent to maintain the enzyme in its insoluble form. Preferably, the enzyme is a protease. In a particularly preferred embodiment, the enzyme is a serine protease. In another particularly preferred embodiment, the enzyme is an alkaline protease. Alternatively, the enzyme is an alpha-amylase.

It is further preferred that the method of the present invention be used for the production of stable, liquid, enzyme-containing detergent formulations. Preferably, the agent for maintaining the enzyme in the insoluble form thereof is a crystallization agent. In a particularly preferred embodiment, the crystallization agent is sodium chloride. If desired, the liquid formulation can be an aqueous formulation, ie, a formulation having a water content. If additionally desired, this liquid formulation may be an aqueous formulation having a water content of 50% (v / v) or greater. In another aspect of the present invention, liquid compositions containing stable enzymes are described. These liquid compositions include a liquid formulation, an insoluble enzyme and an agent for keeping the enzyme insoluble in the insoluble form (crystalline or amorphous) thereof. In such compositions, the insoluble enzyme is maintained in the insoluble form (crystalline or amorphous) thereof, whereby the formation of aerosols (or nebulizations) containing enzymes is substantially avoided. In such formulations, the insoluble enzyme is further maintained in the insoluble form (state) (crystal or amorphous), whereby the enzyme remains stable and retains a substantial portion of its enzymatic activity. The preferred insoluble form of the enzyme is the crystallized form, whereupon crystals of enzymes are formed and provided. Alternatively, the insoluble form of the enzyme may be an amorphous form. In a preferred embodiment, the method comprises the formation of insoluble forms of the enzyme of interest while in the presence of an agent to maintain the enzyme in its insoluble form. In another preferred embodiment, the method comprises the formation of insoluble forms of the enzyme of interest before the combination of the insoluble form of the enzyme with the agent to maintain the enzyme in its insoluble form. Preferably, the enzyme is a protease. In a preferred embodiment, the enzyme is a serine protease. In another particularly preferred embodiment, the enzyme is an alkaline protease. Alternatively, the enzyme is an alpha-amylase. Preferably, the agent for maintaining the enzyme in the insoluble form thereof is a crystallization agent. In a particularly preferred embodiment, the crystallization agent is sodium chloride. It is further preferred that the stable, enzyme-containing liquid compositions of the present invention be stable, liquid, enzyme-containing detergent formulations. If desired, the liquid formulation can be an aqueous formulation, ie, a formulation having a water content. If additionally desired, this liquid formulation may be an aqueous formulation having a water content of 50% (v / v) or greater. These and other additional objects and advantages of the methods and compositions of the present invention will become readily apparent in a reading of the following description, taken in conjunction with the following examples.

Description of the Preferred Modalities A primary aspect of the present invention is that, in liquid compositions, maintaining an enzyme in an insoluble form (crystalline or amorphous) gives the improved storage stability of the enzyme. This is due, at least in part, to the fact that the insoluble form (crystalline or amorphous) of the enzyme is more resistant to denaturation under extreme conditions of pH, heat, storage, etc. This is additionally due, at least in part, to the fact that enzymes (in liquid compositions) in the insoluble (ie crystalline or amorphous) form are less active than those in soluble forms. Accordingly, the stable, enzyme-containing liquid compositions of the present invention are liquid compositions containing enzymes, wherein the enzyme is maintained in its insoluble (ie, amorphous or crystalline) form. In this insoluble form, the enzyme is more resistant to denaturation under extreme conditions of pH, heat, humidity, etc. In this way, the enzyme remains stable and inactive for long periods of time until its use. Such liquid compositions containing enzymes of the present invention include detergent formulations, wherein the enzyme remains stable, to maintain a substantial portion of its enzymatic activity over a long period of time. Stable enzyme-containing liquid compositions of the present invention include an insoluble enzyme (crystallized or amorphous), an agent for maintaining the enzyme in the insoluble form thereof (ie an enzyme crystallization agent) and a liquid formulation in which combine the insoluble enzyme and the agent to maintain the insoluble form of the enzyme. The agent for maintaining the insoluble form of the enzyme can be any agent of this class, which are well known to those skilled in the art. Preferably, this agent is an enzyme crystallization agent. The preferred crystallization agent with which the crystallized enzyme can be combined in liquid formulations according to the teachings of the present invention, can be ammonium sulfate, sodium chloride (U.S. Patent Nos. 5,041,377 and 5,256,557), organic acids (see WO 91/09943 and U.S. Patent No. 5,281,526) and amino acids (see US patent application Serial No. 856,252 issued April 8, 1992). The use of sodium chloride is especially preferred in this regard. The amounts (concentrations) of the agent (crystallization) to maintain the enzyme in the insoluble form thereof, which should be used in the methods of the present invention, to be included in the liquid compositions of the present invention, will be varied as is required by the type and precision of the formulation in which the agent (crystallization) is included to maintain the insoluble form of the enzyme, by the enzyme that is included as well as by the conditions under which the formed composition will be used and / or maintained until use. Such variations can be determined by those skilled in the art. However, it is noted that, it is contemplated herein that the concentration of the crystallization agent to be combined in the final composition will be between 2%. (w / v) of the crystallization agent and about 10% (w / v) of the crystallization agent. It is further contemplated that concentrations of about 5% (w / v) of the crystallization agent will be preferred. Enzymes that can be used in the compositions and methods of the present invention include those enzymes that are capable of being maintained in an insoluble (ie, crystalline or amorphous) form. Preferably, such enzymes are proteases, including serine proteases (such as subtilisins or proteases of the subtilisin type), neutral proteases and alkaline proteases. Examples of such proteases include alkaline proteases that are marketed under the OPTIMASE trademarks (SOLVAY ENZYMES, Inc.) and OPTICLEAN (SOLVAY ENZYMES, Inc.). Other enzymes that are contemplated as being useful in the compositions of the present invention include alpha-amylases, such as those alpha-amylases that are marketed under the trademarks TENASE (SOLVAY ENZYMES, Inc.) and TAKATHERM (SOLVAY ENZIMES, Inc. .), other amylases, lipases, cellulases and / or any other enzyme that is commonly used in detergent formulations (ie for washing clothes or dishes). Also, the use of variants of these enzymes made of proteins, and the combination of different enzyme systems in liquid compositions, are within the scope of the present invention. The precise enzyme (or combination of enzymes) that are included in the compositions of the present invention will vary according to the proposed use of the composition. For example, detergent compositions for washing fabrics, such as clothing, will include alkaline proteases. Enzyme combinations can also be used within the context of the present invention as the circumstances dictate. The amount (concentration) of the enzyme to be used in the methods of the present invention, to be included in the liquid compositions of the present invention, will also be varied as required by the type and precision of the formulation in which it is included. the enzyme, by the agent (crystallization) to maintain the enzyme in the insoluble form thereof, which must also be included and by the conditions under which the composition formed will be used and / or maintained until its use. Such variations can be determined by those skilled in the art. However, it is contemplated herein that the enzyme concentration to be included in the final formulation will be at least about 0.01% (w / v), and more particularly at least about 0.1% (w / v). It is further preferred that the enzyme concentration is at least about 0.5% (w / v). It is much more preferred that the enzyme concentration is at least about 1.0% (w / v). It is also preferred that the enzyme concentration be at least 2.0% (w / v). It is further contemplated herein that the concentration of enzyme to be included in the final formulation will not be greater than about 5.0% (w / v) and, more particularly 2.0% (w / v). It is much more preferred that the concentration of the enzyme is at least about 1.0% (w / v). It is further preferred that the enzyme concentration is not greater than about 0.5% (w / v). It is also preferred that the enzyme concentration is at least about 0.1% (w / v).

The precise insoluble form (physical state) of the enzyme to be included in the liquid compositions of the present invention (ie crystalline or amorphous) will also vary according to the physical state of the formulation and the proposed use thereof. Such variations can be determined by those skilled in the art. The liquid formulations of the present invention in which the insoluble enzymes (crystalline or amorphous) are combined and the agent for maintaining the enzyme in its insoluble form (such as a crystallization agent) can be any liquid formulation. If desired, such formulations may include other additional components, such as surfactants (ie, anionic surfactants) such as linear alkylbenzenesulfonate, or, nonionic surfactants such as alcohol ethoxylate, cationic surfactants or amphoteric ion) as stabilizers (i.e. propylene glycol) ) and pH buffers, to name a few. In this regard, it is noted herein that the principles of the present invention are applicable to liquid formulations having a water content, including aqueous, slurry formulations. In fact, the methods and principles of the present invention are applicable even to those formulations having a water content greater than 50% (v / v). As noted above, the liquid formulations of the present invention can be detergent formulations if desired. In fact, it is contemplated herein that the teachings of the present invention will be particularly applicable to, and useful in, the production, storage and use of liquid detergent formulations. More particularly, it is noted that the teachings of the present invention will be applicable to and useful in the production, storage and use of liquid detergent compositions having a water content. Examples of such commercially available detergent formulations include DOUBLE POWER SURF (Lever Brothers, Inc., E.U.A.), DYNAMO (Colgate-palmolive, Inc., E.U.A.) and STAIN OUT (Colorax, Inc., E.U.A.). The detergent compositions (formulations) of the present invention in which the insoluble enzymes (crystalline or amorphous) are combined and the agent for maintaining the enzyme in its insoluble form (such as a crystallization agent) can be any composition (formulation) of suitable detergent. If desired such formulations may include components such as surfactants (ie, anionic surfactants such as linear alkylbenzenesulfonate, nonionic surfactants such as alcohol ethoxylate, cationic surfactants or amphoteric ion surfactants), stabilizers (ie, propylene glycol), other enzymes (including, but not limited to, amylases, cellulases, peroxidases and oxidases), detergent additives (such as zeolites, diphosphates and triphosphates), additives (such as carbohydrate binders similar to dextrins and cellulose derivatives similar to hydroxypropylcellulose and methylcellulose) and pH buffers (to maintain the detergent compositions at the desired pH thereof), including phosphate or carbon salts. The stable, enzyme-containing liquid compositions of the present invention can be used for various purposes for which they have been formulated. In this respect, the formulations can be used in cleaning compositions for washing clothes, dishes, (for example, in automatic dishwashers), and / or surfaces in need of cleaning with which. In another primary aspect of the present invention, it has been found that the combination, in a liquid formulation, of an appropriate enzyme in the insoluble form thereof (crystalline or amorphous) together with an appropriate agent (such as crystallization agent) for maintaining the enzyme in the insoluble form thereof, provides a liquid composition containing enzymes in which the enzyme shows a remarkable improvement in storage stability. The method of the present invention for the production of the liquid compositions containing enzymes of the present invention includes the formation of enzymes in the insoluble form in question. In this respect, the method can include the crystallization of the enzyme in question. Alternatively, the method can include the formation of the enzyme so that it is in an amorphous form thereof. This insoluble enzyme formation is then followed by the combination in the insoluble form (crystallized or amorphous) thereof with the agent to maintain the enzyme in the insoluble form thereof (such as an enzyme crystallization agent) • to maintain the insoluble structure (crystalline or amorphous) of the enzyme in a liquid formulation. In this form, the stable, liquid, enzyme compositions of the present invention are provided. The crystallization of the enzymes according to the method of the present invention can be carried out by any suitable method, well known to those skilled in the art. Such methods include those described in the US Letters Patent No. 5,256,557, wherein the enzymes are crystallized with the use of amino acids and / or organic acid. Other suitable methods are described in U.S. Patent No. 5,281,526. Enzymes can be formed to be in an amorphous form thereof according to the method of the present invention by any suitable method well known to those skilled in the art. Such methods include those described in US Pat. No. 4,673,647. The combination of the insoluble enzyme (crystallized) and the agent (crystallization agent) to maintain the enzyme in its insoluble form in the desired formulation (including detergent formulations) can be done in different ways. The combination of the insoluble enzyme and the agent can be made by forming the insoluble form of the enzyme in the presence of the agent to maintain the enzyme in the insoluble form thereof (and a liquid formulation, such that one of which and / or in which the insoluble form of the enzyme is formed). In this way, the insoluble enzyme formed is combined (immediately) with the enzyme in the insoluble form thereof in the liquid formulation in which the enzyme is formed in the insoluble form thereof. Alternatively, the combination of the insoluble enzyme and the agent can be made first by forming the insoluble form of the enzyme, followed by the addition of the agent thereto. In this case, the agent can be either in solution (in a liquid composition) when it is added to the enzyme in its insoluble form, so that the insoluble enzyme and the agent combine in the presence of a liquid composition. Alternatively, a liquid composition can be added to a mixture of the enzyme in its insoluble form and the agent, so that the insoluble enzyme and the agent are combined in the presence of a liquid composition. However, it is observed that without considering the precise method selected in all cases, the enzyme in the insoluble form thereof and the agent combine when in the presence of a liquid composition, so that the agent binds the liquid ( water), which keeps the enzyme in the insoluble form of it. The combination of the enzyme in the insoluble form thereof and the agent to maintain the enzyme in the insoluble form thereof should be carried out at temperatures that would not affect the activity of the enzyme (such as for example denaturation of the enzyme). In this regard, it is contemplated herein that temperatures not lower than about 5 ° C and not higher than 50 ° C will be preferred. The combination of the enzyme in the insoluble form thereof and the agent to maintain the enzyme in the insoluble form thereof should be carried out at pHs that would not affect the activity of the enzyme (such as, for example, by denaturing the enzyme). enzyme). In this regard, it is contemplated herein that pHs not lower than about 4.0 and not greater than about 13.0 will be preferred. It is noted herein that the use of the above-described method of the present invention to produce the stable, liquid, enzyme compositions of the present invention significantly reduces the problems associated with the formation of aerosols (nebulizations) containing enzymes. This is achieved by the incorporation of the insoluble (ie crystalline) enzyme in the liquid formulation and by maintaining the insoluble form (ie crystal structure) of the enzyme in the liquid compositions (such as detergent). The present invention is advantageous due to the fact that it provides improved stability (and therefore has an economic impact) and safety (less allergenic). Having thus described, the stable, liquid, enzyme compositions of the present invention, including detergent formulations, as well as the methods of the present invention for the manufacture of such stable, enzyme-containing liquid compositions, including formulations of detergent, the following examples are now presented for illustration purposes and does not mean that they are, and should not be, read as being restrictive.

Example 1 An alkaline protease was obtained from Bacillus alkalophilus and catalyzed following the procedure described, and under the conditions specified in Example 1 of the US Pat. No. 5,256,557. In such a procedure, alkaline protease crystals are formed in the presence of 10% (w / v) sodium chloride (an agent to maintain the enzyme in the crystalline form thereof). In this manner, the stabilized alkaline protease was formed to be in an insoluble (crystalline) form thereof in which it was maintained by the sodium chloride. The concentrate of the crystalline enzyme was then diluted to an activity of approximately 1,000,000 UD (units of Delft) / g by the addition of distilled water (pH 6.5-7.0).

To normalize the solution, sodium chloride was added to it to bring the concentration of sodium chloride of it to 10% (p / v). The following ingredients were also added to the solution to bring their respective concentrations to that which is observed later in the present: boric acid was added to bring the concentration thereof to 1.0% (p / v); calcium chloride was added to bring the concentration of it to 1.0% (w / v); and Corn Syrup Solids were added to bring the concentration thereof to > 50% (w / v) dry solid base (bss). The pH of the solution was adjusted to pH 4.5 with acidic acid (0.1M). In this manner, a liquid composition containing crystalline enzyme, stable according to the present invention, was provided.

Example 2 A 0.1 M borate buffer solution (pH 8.5) comprised of boric acid (0.1 M) and sodium borate (0.1 M) in water was prepared.

The liquid composition containing crystalline enzymes, stable containing 10% (v / v) of enzyme crystallization agent (sodium chloride) was then added (in a ratio of 1: 1 (v / v)) to the borate buffer, so that a liquid composition containing crystalline enzymes was formed, stable containing 5% (v / v) of enzyme crystallization agent (sodium chloride). The alkaline protease of this composition had an initial activity of approximately 500,000-800,000 UD / g. A liquid formulation of the same alkaline protease (initial activity of 1,000,000 UD / g) formulated in propylene glycol, sold under the name OPTICLEAN L-1000 (SOLVAY ENZYMES, Inc.) Was obtained. The respective samples of both the liquid composition of the present invention having the crystalline form of this alkaline protease and the liquid composition of the alkaline protease formulated in propylene glycol were then incubated at 50 ° C at pH 8.5 until they were removed at selected intervals of time (0, 3, 6, 12, 24 and 38 hours) and the activity of the alkaline protease of the same measurement according to the following described procedure, and under the specific conditions of Example 1 of the US Patent No. 5,256, 557 The results of these trials are summarized in Table 1.

Table 1 Time (Hr) of Percent remaining activity incubation at 50 ° C, pH 8.5 Liquid Crystals 0 100 100 3 71 95 6 60 80 12 45 83 24 20 80 38 7.5 65 As can be seen in Table 1, the crystalline (insoluble) form of the alkaline protease is more stable at a high pH and high temperature than the same alkaline protease that was in the soluble form.

Example 3 The aqueous stability of the alkaline protease in the liquid form (OPTICLEAN L-1000) and the stabilized crystalline alkaline protease crystals of Example 1 were then compared at different pHs. The stable, crystalline enzyme-containing liquid composition containing 10% (w / v) of enzyme crystallization agent (sodium chloride) of the Example 1 was then added (in a 1: 1 ratio (v / v)) to the borate buffer as described in Example 2, so that a stable crystalline enzyme-containing liquid composition containing 5% (w / v) of enzyme crystallization agent (sodium chloride) was formed. sodium). The alkaline protease of this liquid composition had an initial activity of approximately 5,000,000-800,000 UD / g. OPTICLEAN L-1000 was obtained as described above in Example 2. The respective samples of both the liquid composition of the present invention having the crystalline form of this alkaline protease and the liquid composition of the alkaline protease formulated in propylene glycol, then they were incubated at different pHs (ie pH 4 at pH 10.0) at 50 ° C for 24 hours, at which time the samples were removed and the activity of the alkaline protease thereof was measured following the procedure described, and under the conditions specified in Example 1 of U.S. Patent No. 5,256, 557. The results of these tests are summarized in Table 2.

Table 2 pH of the remaining activity percentage incubation 50 ° C, 24 hours Liquid Crystals pH 4.0 27.5 36.5 pH 6.0 28.0 37.4 pH 8.0 37 51.0 pH 9.0 20 32.0 pH 10 OO 14 30.0 Here again, the results of Table 2 confirm that the crystalline (insoluble) form of the enzyme is more stable than the enzyme in the soluble form.

Example 4 The storage stability of the liquid form of the alkaline protease was then compared to the stable crystallized alkaline protease formulation of the present invention in an aqueous thick solution detergent formulation. The stable, crystalline enzyme-containing liquid composition containing 10% (v / v) of enzyme crystallization agent (sodium chloride) was obtained as described above in Example 1. The crystals were then concentrated to approximately 5,000,000 UD / g using microfiltration as also described in the Example 1 of the North American patent No. ,256, 557. Then, a part (w / w) of glass concentrate (100 grams) was mixed with four parts (p / p) of a thick suspension detergent base (400 grams) to produce alkaline protease crystals in the detergent base with an activity equivalent to the protease activity of OPTICLEAN L-1000 (1,000,000 UD / g).

The aqueous detergent slurry formulation that was tested herein is the commercially available formulation known as DOUBLE POWER SURF (Lever Brothers, Inc., E.U.A.). This formulation contains a liquid, active enzyme. Therefore, prior to the addition of the alkaline proteases to be tested to the detergent formulation, the enzyme was previously inactivated in this detergent formulation by heating the thick detergent solution at 60 ° C until all the active enzyme was destroyed. (10 minutes) . This thick, aqueous, remaining suspension detergent formulation was then used as the base detergent formulation to test the stability of the formulations of the invention. The OPTICLEAN L-1000 liquid was obtained as described in Example 2. The respective samples of the liquid detergent composition containing crystalline, stable enzymes of the present invention and the OPTICLEAN L-1000 were then kept at 37 ° C until the individual samples thereof were removed at different time intervals (as specified in Table 3) and the activity of the residual protease was measured following the procedure described, and under the conditions specified in Example 1 of the North American patent No 5,256,557. The results of these tests are summarized in Table 3.

Table 3 Sample # Description Percent of the formulation loss of protease activity 2 weeks 4 weeks 1 liquid enzyme 11 31 formulated in propylene glycol (1, 000, 000 UD / g) 2 Crystalline enzyme 0 formulated on the basis of thick suspension detergent (1, 000,000 UD / g) The remarkable improvement in storage stability of the alkaline protease crystals in thick suspension detergent formulations could be due to the maintenance of the crystal structure of the enzyme by the presence of a sufficient amount of enzyme crystallization agent in the formulation, that is, NaCl (from crystal concentrate). The microfilter concentrate of the alkaline protease crystals contained 10% (w / v) of sodium chloride. The detergent slurry formulation containing 20% (w / v) concentrate of the alkaline protease crystals microfilter contained 2% (w / v) of alkaline protease crystals during storage.

Example 5 The aqueous suspension detergent slurry formulation (DOUBLE POWER SURF) was obtained and heat treated (to inactivate enzymes thereof) as described above in Example 4. Liquid OPTICLEAN L-1000 was obtained as described in Example 2. The stable, crystalline enzyme-containing liquid composition containing 10% (w / v) of enzyme crystallization agent (sodium chloride) was obtained as described in Example 1. To the 99-gram samples , respective of the thick suspension detergent base, either one gram of liquid alkaline protease (1,000,000 UD / g) or one gram of the liquid composition containing stable crystalline enzyme was added. The resultant aqueous slurry detergent formulations were mixed for a uniform concentration. After checking the activity of the initial enzyme, these samples were stored at 37 ° C. The samples were periodically removed (as discussed later in Table 4) and activity was measured, following the procedure described, and under the conditions specified in. Example 1 of U.S. Patent No. 5,256,557. The results of these tests are summarized in Table 4.

Table 4 Sample # Description Percent of formulation loss of protease activity Weeks at 37 ° C 2 weeks 4 weeks liquid enzyme 52 58 formulated in a thick suspension detergent base Crystal enzyme 15 26 formulated on detergent base thick suspension The results observed with reference to table 4 showed that a rapid inactivation of the enzyme occurred when the alkaline protease was not stabilized by sodium chloride. The loss of enzyme activity in the detergent-based slurry containing alkaline protease crystals that have been combined with the sodium chloride crystals is considerably less than the loss of activity in the detergent base containing crystals of alkaline protease that have not been combined (stabilized with sodium chloride). It should be noted that, while the results of Table 4 show that the stabilized crystalline enzyme used in the slurry detergent formulations had a loss of activity, it is believed that this loss could be due to the slow resolubilization of the crystals in the The formulation of thick suspension detergent due to the lower concentration of sodium chloride in the formulation. In this way, the improved activity of the enzyme (s) in the detergent formulation was achieved by the incorporation of enzyme in the crystalline form with an appropriate concentration of enzyme crystallization agent. Modifications of the stable, enzyme-containing liquid compositions of the present invention and methods for the manufacture thereof can be made without departing from the basic spirit of the present invention. Accordingly, it will be appreciated by those skilled in the art that, within the scope of the appended claims, the invention may be practiced differently than has been specifically described herein.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the content of the following claims is claimed as property.

Claims (20)

1. A method for the production of a liquid composition containing enzymes, stable, characterized in that it comprises: the formation of an enzyme, so that the enzyme is in an insoluble form thereof and the combination, in a liquid formulation, of the enzyme in the insoluble form thereof with an agent to maintain the enzyme in the insoluble form thereof, thereby forming a stable liquid composition containing enzymes.

2. The method according to claim 1, characterized in that the formation of the enzyme in the insoluble form thereof is comprised of the crystallization of the enzyme, so that the enzyme has a crystalline form.

3. The method according to claim 2, characterized in that the combination, in a liquid formulation, comprises combining, in a liquid formulation, the crystalline enzyme with an enzyme crystallization agent to maintain the enzyme in the crystalline form thereof, with which forms a liquid formulation containing enzymes, stable.

4. The method according to claim 3, characterized in that the crystallization agent is sodium chloride.

5. The method according to claim 1, characterized in that the enzyme is a protease.

6. The method according to claim 1, characterized in that the enzyme is a serine protease.

7. The method according to claim 1, characterized in that the serine protease is an alkaline protease.

8. The method according to claim 1, characterized in that the liquid formulation is an aqueous formulation, whereby an aqueous composition is formed.

9. The method according to claim 1, characterized in that the liquid formulation * is a detergent formulation.

10. A method for the production of an aqueous detergent composition containing enzymes, stable, characterized in that it comprises: the crystallization of an enzyme, thereby providing an enzyme having a crystalline form and the combination, in an aqueous detergent formulation, of the crystallized enzyme and an enzyme crystallization agent to maintain the enzyme in the crystalline form thereof, whereby an aqueous detergent composition containing enzymes is formed, stable.

11. A liquid composition containing enzymes, stable, characterized in that it comprises: at least one enzyme having an insoluble form thereof, an agent for maintaining the enzyme in the insoluble form thereof and a liquid formulation, wherein the enzyme in the Insoluble form thereof and the agent are combined so that the enzyme is stable and the enzymatic activity thereof is retained.

12. The liquid composition containing enzymes according to claim 11, characterized in that the enzyme in the insoluble form is a crystalline enzyme.

13. The liquid composition containing enzymes according to claim 12, further characterized in that the agent for maintaining the enzyme in the insoluble form thereof is a crystallization agent.

14. The liquid composition containing enzymes according to claim 13, characterized in that the crystallization agent is sodium chloride.

15. The liquid composition containing enzymes, stable according to claim 11, characterized in that the enzyme is a protease.

16. The liquid composition containing enzymes, stable according to claim 11, characterized in that the enzyme is a serine protease.

17. The liquid composition containing enzymes, stable according to claim 11, characterized in that the serine protease is an alkaline protease.

18. The liquid composition containing enzymes, stable according to claim 11, characterized in that the liquid formulation is an aqueous formulation, whereby an aqueous composition is formed.

19. The liquid composition containing enzymes, stable according to claim 11, characterized in that the liquid formulation is a detergent formulation, whereby a detergent composition is formed.

20. A liquid composition containing enzymes, stable, characterized in that it comprises: at least one crystallized enzyme, an agent for maintaining the enzyme in the crystallized form thereof and an aqueous detergent formulation, wherein the crystallized enzyme and the agent are combined, so that the enzyme is maintained in the crystalline form thereof and the enzymatic activity thereof is retained.