COMPOSITIONS OF CELLULOSE TREATED WITH PROTEASE AND
PURIFIED, AND METHODS TO REDUCE THE RETRO-DYED
DURING ENZYMATIC WASHING WITH STONES
Field of the Invention This invention relates to compositions and methods for reducing or preventing back-staining of indigo blue dye on denim during washing with fabric stones and denim garments using cellulase enzymes. BACKGROUND OF THE INVENTION Denim is a woven cotton fabric in which the warp, usually blue, has been dyed with the indigo dye. A desirable feature of indigo-dyed denim fabric is the appearance created by alternating blue and white filaments of the warp and weft yarns, which upon occurrence of normal wear and wear give the denim a white-on-blue appearance. A popular view of denim is the sight washed with stones. This view washed with stones consists of a blue color generally lighter than unwashed denim, with localized areas, particularly around the seams, even lighter in color. Stone washed material often has a softer texture and maintains the desired contrast of white on blue. Traditionally, stone washing has been carried out by washing the denim material in the presence of pumice, which results in a fabric having a worn or used appearance and the desired white-on-blue contrast described above. Enzymes, particularly cellulases, are currently used in denim processing. In particular, cellulases are used to give a stone-washed appearance without the need for such a high pumice load as that used in traditional stone washing. This processing method is referred to herein as enzymatic "stone washing", even if there are no stones present in the washing machine. The use of enzymes for stone washing has become increasingly popular because the use of stones has several disadvantages. For example, the stones used in the process cause wear and tear on the machinery, environmental waste problems due to the sand grains produced and result in high labor costs associated with the manual removal of stones from the machines and the garment bags. Accordingly, the reduction or removal of stones in the wash may be desirable. Contrary to the use of pumice stones, enzymes
(particularly cellulases) are safe for the machine, result in little or no waste problem, and drastically reduce labor costs. Therefore, it can be beneficial to use enzymes for stone washing. However, even when the use of enzymes such as cellulase can be beneficial in comparison with stones, there are some problems associated with the use of enzymes for this purpose. For example, a problem with some cellulases, such as the cellulases of the fungus that rots Trichoderma roots, is what can be described as "re-deposition" or "retro-staining" (both terms are used interchangeably herein) of some of the dye back on the fabric during the enzymatic stone washing process. Such re-deposition or retro-dyeing leads to a blue coloration on the white threads of the denim, resulting in less contrast between the blue and white threads and the points of abrasion (ie, a view of blue on blue instead of the preferred view of white on blue). See American Dyestuff Repórter, September 1990, pp. 24-28. Re-depositing or retro-dyeing is objectionable for some users. For example, although Trichoderma cellulases exhibit a much higher specific activity on the denim material than Humicola cellulases, Humicola cellulases are often preferred due to their lower level of retro-dyeing. This is true, even though the much higher potency of the Trichoderma cellulase allows the use of smaller amounts of enzyme to achieve a higher degree of abrasion in considerably shorter processing times. The problem of re-depositing the dye during stone washing has been a concern for those who process denim. Previous attempts to address the problem with Trichoderma cellulase compositions include the addition of additional chemicals against re-deposition, such as surfactants or other agents, in the washing of cellulase to help disperse the loose indigo dye and reduce re-deposition. In addition, those who process denim have tried to use cellulases with less specific activity on the denim, along with additional rinses. This results in additional costs of chemicals and longer processing times. Another method that attempts to address the problem of re-deposition includes adding a faint bleaching agent or dye removal agent in the process. This method affects the final tone of the garment and increases the processing time. Although these methods help to a limited degree in reducing re-deposition, the methods are not entirely satisfactory and some objectionable retro-dyeing remains. The use of enzymes and stones together can be advantageous to reduce the degree of re-deposition; however, it leaves the processor some of the problems associated with the use of stones alone. Another method, as described by Clarkson et al. In PCT publication No. WO 94/29426 (hereinafter "Clarkson et al."), Has been able to include a protease enzyme added in the stone wash treatment. It was found that denim treatment with a composition comprising a re-deposition cellulase and an added protease improves the contrast between the white and blue threads and reduces the re-deposition of dye. Acting on the washing machine, it is thought that proteases prevent cellulase proteins from binding colored particles back onto the surface of the denim and, even when used sparingly, do not have a severe negative effect on the sight resulting from abrasion. resultant caused by the action of the cellulase. This method, while providing some advantages, is expensive and requires careful control because proteases, by their very nature, tend to destroy cellulase enzymes. The technician must play with the balance between the desirable proteolytic effect by reducing the retro-dyeing and the undesirable proteolytic effect by reducing the activity of the cellulase. In the Clarkson et al. Process, the protease is used essentially as a stain remover or stain inhibitor and must be included in the washing process. Clarkson et al. Teach three options: (1) add the protease directly to the washer with the cellulase enzyme, (2) add the protease to the rinse cycle after a cellulase treatment, or (3) physically mix the protease with the cellulase before washing Regarding the basic process of adding the protease and the cellulase together, adding the protease to the rinse cycle prevents considerable proteolytic attack on the cellulase, but has the disadvantage of adding an additional processing step. The physical pre-mixture of cellulase and protease, on the other hand, allows a simple, easy-to-use formulation, but results in a difficult balance between the desirable proteolytic effect by removing the dye and the undesirable proteolytic effect by destroying the activity of the cellulase. For example, a highly active protease can completely destroy the cellulase enzyme during the normal time it requires for storage and shipment. This problem of shelf stability can be handled, but requires: (1) selection of a protease that has good anti-staining power, but can digest cellulase, when more, to a limited extent (the preferred choice is subtilisins, which they are not highly active against cellulase but are well known as potent dye removers), and (2) a pre-incubation of selected protease and cellulase at an elevated temperature to ensure that the proteolytic attack present on the cellulase is carried to completion and that a commercial formulation will be stable during storage and shipment. The action of more potent proteases, particularly the papain protease on cellulases of Trichoderma, has been investigated extensively. It has been found that limited amounts of papain digestion can divide the nuclear domains of Trichoderma cellobiohydrolases from their natural binding domains. This has the effect of essentially eliminating any measurable activity these enzymes have against crystalline cellulose, such as Avicel, or cotton, while still retaining their activity against soluble substrates such as beta-glucan. As a result, previous researchers concluded that the natural ligation domain plays a critical role in enabling the attack of the cellobiohydrolase enzyme on crystalline cellulose. The degree of treatment necessary for the papain to completely eliminate CBH activity on crystalline cellulose was roughly 0.1 to 0.5 grams of the protein papain per gram of cellulase protein, multiplied by the treatment time in minutes (g min / g), that is, the weight ratio of the protease protein to the cellulase protein multiplied by the treatment time. Based on the limitations of the methods previously tried to reduce or prevent re-deposition, there is a need for more easily controlled and more cost-effective methods to address the issue of re-deposition or retro-dyeing. of the dye during the stone wash treatment. Accordingly, it would be desirable to find an enzyme composition or method that is effective from a cost perspective, has good shelf stability, high potency, and does not include a re-depositing or re-staining cellulase. SUMMARY OF THE INVENTION The inventors of the present invention ("the inventors") have found that washing indigo-stained cotton denim with a low retro-dyeing cellulase enzyme composition, done by subjecting a Trichoderma cellulase containing the enzymes cellobiohydrolase and endoglucanase at a limited proteolysis (ie, a limited protease treatment) and the subsequent removal of the added protease, is an improvement over the use of a re-depositing cellulase preparation or one that includes both a cellulase as a protease. The denim produced by the treatment with such composition unexpectedly has a reduced level of dye re-deposition and therefore good contrast between the blue and white threads in the denim. The composition is shelf stable, requires considerably less protease than previous methods, and has a surprisingly low level of re-deposition even when the "retro-staining inhibitory composition" (ie, the protease) taught as required by Clarkson and collaborators. The inventors have also provided methods to recover and recycle protease so that this expensive ingredient can be reused. In the denim washing process, a small percentage of surface-active chemical surfactant may optionally be added to the compositions or methods described herein. If a surfactant is added, it can be added either with the cellulase in the wash or as a post-treatment rinse. In addition, the denim washing process can be carried out with or without stones added to the compositions described herein. Detailed Description of the Invention Denim that is washed with stones with a cellulase enzyme composition of Trichoderma that has been subjected to a limited protease treatment and subsequent purification to remove the protease, shows a dramatic reduction in the level of retro-dyeing and a visible increase in the contrast between white and blue threads. Although the inventors do not wish to be limited by any particular theory, a possible explanation for the seemingly contradictory observations that, on the one hand, protease is required in the washing machine (Clarkson et al.) And, on the other hand, that it does not need to be present ( this invention), is that there are two separate and distinct mechanisms by which proteases can affect back-staining in a denim washing process. The first mechanism is that described by Clarkson et al, where the protease simply acts as a dye removal agent in the washing machine. This mechanism is consistent with Clarkson's findings that proteases can remove the re-deposited dye even after it has stained white jean in a previous treatment with cellulase. It is also consistent with the well-known use of proteases as stain removers in detergent systems. It is not surprising that the dye-based stains created by the cellulase proteins can be removed by one of the well-known approaches to treat spots related to proteins: proteases. Nevertheless, under this first mechanism, the technicians in the matter would not expect that the proteases would be able to mitigate the retro-dyeing in the washing machine if they are never put in the washing machine. To explain the inventors' surprising discovery that proteases seem to do just that, the inventors suggest that there is a second mechanism that is more subtle and less obvious than the first. In particular, the inventors believe that a limited treatment with protease changes the mode of action of the cellulase enzymes, making the small particles easily dispersed do not retro-dye. Although it has been reported that proteases can cause the cellobiohydrolase components present in the complex of the cellulase enzyme of Trichoderma to be inactive against crystalline cellulose by breaking their natural ligation domains with cellulose, the inventors hypothesize that such treatment can still leave the cellobiohydrolase enzymes capable of making small cuts in cellulose which are not detectable by themselves but which, when used in combination with other components in the complex of the cellulase enzyme of Trichoderma, ie the endoglucanases, lead to substantial abrasion in a denim wash environment. The inventors further suggest that, because the modified cellobiohydrolases do not have binding domains, their action against crystalline cellulose is feasible to be less localized and more evenly distributed over the denim fibers than under treatment with the intact Trichoderma cellulase. For intact Trichoderma cellulase enzymes, their highly localized mode of action can lead to relatively large particles being released from the main cellulose body as enzymes cut directly through large portions of the fiber. In contrast, the more distributed pattern of action of Trichoderma cellulase compositions treated with protease can lead to smaller, more easily dispersible particles that become loose from the cellulose main body in an environment where there is a considerable amount of shear or mixed. As a result, it would have less retro-tinted. This second mechanism, although not yet recognized, was probably playing a minor role in the combined cellulase and protease treatments described by Clarkson. It went unnoticed because previous researchers focused on exploiting the most obvious use of the protease to remove stains in the washer. It is not fully exploited because, if it is planned to make a single commercial composition of enzymes containing both cellulase and protease enzymes, it is difficult to operate the proteolysis reaction effectively. In Clarkson's own words, there is a difficult compromise and "balance between the proteolytic effect by reducing the retro-dyeing and the proteolytic effect by reducing abrasion." In this way, in order to make a composition of combined cellulase / protease enzymes that can be added directly to a washing machine, those skilled in the art certainly want to avoid the use of proteases such as papain, which are known to destroy the activity on crystalline cellulose in others more known for their anti-dyeing properties in detergents, for example, subtilisin. Surprisingly, the inventors have found that better results are achieved (1) by abandoning the benefits of this first "anti-dyeing" mechanism (i.e., removing the protease from the enzyme preparation and thereby denim washing), and (2) taking advantage of this change to modify the conditions of the proteolytic reaction to obtain a more intense and more aggressive treatment and thereby maximize the impact of the second previously unrecognized mechanism. All in all, the new compositions that result from this approach allow a superior denim wash and more cost effective. Before discussing this invention in greater detail, the following terms will be defined. The term "Trichoderma cellulase composition" comprises at least one or more of the cellobiohydrolase enzymes (CBH), and one or more of the endoglucanase (EG) enzymes produced by the fungal microorganism Trichoderma sp. When the composition is produced by a natural Trichoderma microorganism, and each of these components is found in the proportion naturally produced by the microorganism, the composition is sometimes referred to herein as a "complete or natural Trichoderma cellulase composition". It is contemplated that the Trichoderma cellulase compositions of the present invention may also refer to any cellulase composition that contains both a cellobiohydrolase and endoglucanase that are obtained from Trichoderma sp. which has been genetically engineered so as to overproduce, byproduce or not produce one or more of the CBH and / or EG cellulase components. These endoglucanases and cellobiohydrolases can include not only enzymes that are a part of the natural Trichoderma cellulase composition, but also such modified cellulase compositions as truncated cellulase proteins that comprise either the binding domain or the core domain of the CBHs. or EGs, or a portion or derivation thereof. Other examples of modified cellulase compositions may include alterations in the degree of glycosylation or substitution of amino acid (s) in the primary structure of the cellulases or truncated cellulases. It is also contemplated that any natural or modified versions of the natural Trichoderma cellulases, such as those noted above, should be considered as Trichoderma cellulase compositions even if they are produced in a genetically modified host microorganism other than Trichoderma. The term "Trichoderma cellulase treated with protease" refers to a Trichoderma cellulase composition in which a significant fraction of the CBH core domains have had their CBH ligation domains cut, for example by treatment with an added protease enzyme. However, the Trichoderma cellulase compositions treated with protease should not have any significant residual or incremental active protease over the amount that is naturally produced by the microorganism. Such incremental amount should be, for example, less than 0.1% of the total amount of protein in the cellulase enzyme composition. It is contemplated that the protease-treated cellulase compositions of the present invention may include both preparations in which an added protease enzyme is used to cut the CBH core and ligation domains and modified cellulase compositions in which the CBH core domain it is produced directly without its ligation domain by means of a genetically modified microorganism. In all cases, notwithstanding the above, a Trichoderma cellulase composition treated with protease must contain endoglucanase activity as well as the core protein CBH. The methods of the present invention comprise contacting denim to be washed with stones in partial or complete enzymatic form with a Trichoderma cellulase composition treated with protease in an amount sufficient to achieve the desired level of dye removal from the garment. . The use of such an enzyme will result in a garment with excellent contrast between the blue and white threads and a low level of retro-dyeing. The enzyme itself will have good stability and will not be in danger of considerable degradation of protease. In one embodiment of this invention, the Trichoderma cellulase composition treated with protease is produced by contacting a Trichoderma cellulase composition with an added protease enzyme, where the degree of treatment, as defined by the weight ratio of the protease protein to the cellulase protein, multiplied by the average treatment time, is in the range of between 1.0 and 10,000 g min / g. The protease is then removed from the cellulase using a chromatographic separation. In a further embodiment of this invention, this Trichoderma cellulase composition treated with protease is added to a denim washing machine dyed with indigo dye and used to create an abrasion appearance, with high contrast between the blue and white fibers. of the denim. Cellulase Enzymes Trichoderma cellulase compositions are typically produced in submerged culture of the Trichoderma fungus, and methods for their production and recovery are well documented in the literature and are well known to those skilled in the art. Commercial sources of these enzymes include Iogen Corporation, Genencor International, Novo Nordisk, Gist-Brocades, Sigma Chemicals and Enzyme Development Corporation. One of the preferred Trichoderma cellulase compositions of this invention is that produced by strains of the fungus Trichoderma longibrachiatum in which the relative concentrations of the enzymes CBH1, CBH2, EG1, EG2 and EG3 are all essentially consistent with those found in a Complete or natural Trichoderma cellulase composition. Commercial cellulase preparations are not 100% cellulase protein and often include fillers, buffers, stabilizers and other ingredients. The total cellulase protein can be measured by means of various assay methods known in the art. The assay used is preferably the commercially available Biorad Coomassie Blue Protein assay sold by Biorad Company, Los Angeles, California, USA, using highly purified cellulase protein as a standard. Protease Added Proteases are available from various sources, including microbial, plant and animal sources, and are documented in the literature. Some important microbial proteolytic sources include Bacillus licheniformis, Bacillus subtilis, and Aspergillus oryzae. Important sources of plant proteases include papaya for papain and pineapple for bromelain.
Proteases suitable for the invention include serine, cysteine, aspartic acid and metallo proteases. One of the preferred proteases is the cysteine protease papain. The proteases are commercially available in a timely manner from firms such as Sigma Chemicals in several different forms, including liquid solutions, powders or as insoluble enzymes attached to solid supports. In a preferred embodiment of this invention, the papain protease is used in a liquid suspension. Commercial protease preparations are not 100% protein protease and often include fillers, buffers, stabilizers and other ingredients. The total protease protein can be measured by means of various assay methods known in the art. The assay preferably used herein is the commercially available Biorad Coomassie Blue Protein assay, sold by the Biorad Company of Los Angeles, California, United States. Protease Treatment The protease-limited treatment of this invention comprises contacting a liquid Trichoderma cellulase composition with an added protease under controlled reaction conditions for a defined period of time. A person skilled in the art will recognize that the appropriate measure of treatment will depend on the temperature, the pH, the concentration chosen to prepare the mixture and the specific activity of the protease enzyme that has been selected, and will recognize, in addition, that routine procedures of test to select an optimal set of process conditions for a given cellulase and protease composition given. In a preferred embodiment, the limited treatment with protease is carried out at an elevated temperature between 20 and 60 ° C, and more preferably between 30 and 50 ° C. In the most preferred embodiment, a temperature of about 37 ° C was used. In a preferred embodiment, the protease-limited treatment is carried out at a pH between 3.0 and 8.0, and more preferably between 4 and 7. In the most preferred embodiment, the pH is between 4 and 5. In a preferred embodiment, the protease-limited treatment is carried out with a concentration of the cellulase protein between 5 and 250 g / l and, more preferably, between 50 and 200 g / l. In the most preferred embodiment, the concentration of the cellulase protein is around 100 g / l. In a preferred embodiment, the limited treatment time with protease is between five minutes and four weeks, and more preferably between 1 and 120 hours. In the most preferred embodiment, where the protease is papain, the treatment time is around 24 to 48 hours. In a preferred embodiment, the degree of treatment, as defined by the weight ratio of protease protein to cellulase protein, multiplied by the average treatment time, is in the range of between 1.0 and 10,000 g min / g , and more preferably between 10 and 1,000 g min / g. In the most preferred embodiment, where the protease is papain, the degree of treatment is in the range of about 200 g min / g. Under these preferred conditions, this means that the concentration of papain is around 14 g / l. A preferred method of tracking the progress of a proteolysis reaction is to use filter paper and CMC assays that measure, respectively, FPUs and CMCUs of cellulase activity (Ghose 1987). Preferably, the protease treatment would be run to a degree that the cellulase loses at least 5% of its initial activity measured in filter paper units (FPUs) and not more than 50% of its initial activity as measured in CMC units . Even more preferably, the cellulase must be treated to a degree where it loses at least 10% of its initial cellulase activity, as measured in FPUs, and maintains substantially between 70 and 100% of its initial activity as measured in CMCUs. Even more preferred, the cellulase must be treated to a degree where it loses about 50% of its initial cellulase activity, as measured in FPUs, and less than 10% of its initial activity, as measured in CMCUs. Removal and Recovery of Protease The method of this invention further requires that the proteolytic reaction be stopped when it has reached the desired degree of treatment, such that considerable amounts of exogenous protease do not contaminate the enzyme composition of Trichoderma cellulase treated with protease of this invention. The reaction can be stopped, for example, by sudden cooling or adjusting the pH. The added protease can then be separated from the cellulase complex. One skilled in the art will recognize that there are several means to selectively remove added protease from a Trichoderma cellulase enzyme composition, including chromatographic separation, selective precipitation, ultrafiltration or filtration (if an insoluble enzyme is used on a solid support). The appropriate method of removal will depend on the specific nature and form of the protease that has been selected for treatment. A preferred method of achieving this separation is to bind a dissolved protease to a solid material and then wash the cellulase therefrom. A similar ligation medium, used herein, is a commercially available cation exchange resin, S-Sepharose, sold by Pharmacia Biotech, Uppsala, Sweden, which will bind many commercial protease enzymes when contacted with a solution of cellulase and protease at a pH below 6.0. A preferred method of using S-Sepharose, which is applicable to remove the papain protease from a cellulase enzyme preparation of Trichoderma, is to dialisize a mixture of cellulase and protease at a conductivity of 3,000 μS or less and then pass it over a resin S-Sepharose at a pH between 4.5 and 5.0 and at a temperature below 20 ° C. In a preferred embodiment, the protease is recovered and reused in another Trichoderma cellulase load. A preferred method of recovering the protease is to bind it to an S-Sepharose resin at a pH between 4.5 and 5.0 and a temperature below 20 ° C. The S-Sepharose resin can bind approximately 100 g / l of the papain protease. The mixture of cellulase and papain is passed over the resin and when it is fully loaded with papain, it is washed with softened water to remove any cellulase that contaminates and then washed with a 1 M sodium chloride solution to desorb the papain. The papain solution is then dialyzed to remove the excess salt and is then ready to be reused. During this recovery operation, it is important to maintain a reducing environment because papain is subject to reversible oxidative inactivation. Product Formulation The cellulase compositions of this invention may also comprise various adjuvants known to those skilled in the art. For example, a surfactant (anionic or non-ionic) compatible with the cellulase composition would be useful in the compositions of the present invention. Preferred surfactants are non-ionic, such as the polyoxyethylated alcohols found in the Triton (trademark) series of surfactants (octylphenoxypolyethoxyethanol nonionic surfactants) which are commercially available from Union Carbide. It should be noted that the inclusion of a surfactant can further improve the relative contrast between the white and blue yarns and reduce the amount of colorant re-deposition. Other materials may also be used with or placed in the composition, as desired, including stones, fillers, solvents, buffers, enzyme stabilizers, pH control agents, enzyme activators, edifiers, other anti-re-depositing agents and the like. . The enzyme composition can be formulated as a solid product where the solid can be granular, spray dried or agglomerated. Alternatively, the enzyme composition can be formulated as a liquid, a gel or a paste product. A liquid preparation is preferred herein. Denim Washing Denim washing to create a "stone washed" appearance can be achieved substantially by using a stone or stone-free process in which denim or denim is mechanically stirred in a washing machine with an aqueous composition that contains the cellulase compositions of Trichoderma treated with protease. The amount of the composition used to treat denim would depend on the concentration of the cellulase protein in the cellulase composition, the amount of the denim substrate in the washing machine, and the desired amount of stone washing effect, and other well-known parameters for the technicians in the matter. The preferred amount of the Trichoderma cellulase composition treated with protease is generally between 500 and 200,000 CMC units of enzyme per kilogram of denim, and more preferably between about 5,000 and 100,000 CMC units per kilogram of denim. In a preferred embodiment, the denim washing treatment is carried out at an elevated temperature between 30 and 70 ° C and more preferably between 45 and 55 ° C. In a preferred embodiment, the denim wash treatment is carried out at a pH between 4.0 and 7.5, and more preferably between 4.5 and 6.5. In the most preferred embodiment, the pH for the denim treatment is around 6.0. In addition to the cellulase composition, the denim washing step can also use a variety of other process aids. For example, it would be useful to add to the washer a surfactant (anionic or non-ionic) compatible with the cellulase composition, in the methods of the present invention. Preferred surfactants are non-ionic surfactants, such as polyoxyethylated alcohols found in the Triton (registered trademark) series of surfactants (Octylphenoxypolyethoxyethanol nonionic surfactants), commercially available from Union Carbide. It should be noted that the inclusion of a surfactant can further improve the relative contrast between white and blue yarns and reduce the amount of colorant re-deposition. Other materials can also be used with or placed in the washing machine, as desired, including stones, fillers, solvents, buffers, enzyme stabilizers, pH control agents, enzyme activators, edifiers, other anti-re-depositing agents, and Similar. The plies The foregoing description provides a discussion of the compositions of the invention and the methods of making and using the compositions in the "stone wash" of cloth articles of clothing. The following examples provide specific details regarding the compositions and methods of the invention. It will be apparent to those skilled in the art, based on the teachings contained herein, other options of added protease and cellulase, as well as washing conditions such as concentration, measurement, pH, temperature and the like. Example 1 Preparation of a Trichoderma Cellulase Composition Treated with Protease Approximately 600 liters of a natural Trichoderma cellulase preparation was produced by fermentation of Trichoderma longibrachiatum and dialysed at a conductivity of 450 μS. Although this product was not stabilized or preserved, it is available in a stabilized and conserved form as logen cellulase from logen Corporation. A substantially similar material can be prepared by simply diallylating logen cellulase to remove stabilizers and preservatives. It was then concentrated to a volume of 500 liters by ultrafiltration. The resulting dialysis product has a protein concentration of 140 g / l and an endoglucana-sa activity of 1, 599 CMC units / ml using the method of Ghose (1987). 150 liters of the preparation were removed and the remaining preparation was then mixed with 150 liters of soft water and 40 kg of papacon powder Biocon, available from Quest International
(product number 5x98490) and having a papain concentration of 105 g / kg and an activity of 1,000 milk coagulant units (MCU) / mg of papain powder, as specified by Quest International. The pH was adjusted to 4.8 using sodium benzoate and the mixture was incubated roughly at 35-40 ° C for 42 hours. The resulting treatment degree was approximately 216 g min / g. 49% of the initial cellulase activity was lost during this protease treatment, as measured in FPUs, and less than 10%, as measured in CMCUs. The mixture was then quenched at 20 ° C for a period of one hour. The mixture was then rinsed in a dish pre-coated with diatomaceous earth and a frame filter. With rinse water, the preparation was diluted to a volume of about 960 liters. The protease was removed from the protease and cellulase mixture, passing the preparation on a cation exchange resin S-Sepharose, according to the resin manufacturer's instructions (Pharmacia technical manual 18-1022-19"Ion Exchange Chromatography: Principles and Methods ", 1991). S-Sepharose was first equilibrated to a pH of 4.7 with acetate buffer; subsequently, the protease / cellulase mixture was loaded onto the resin to a measure of approximately 13 g of papain protein per liter of packed resin. The resin was washed with acetate buffer, pH 4.7. The combined effluents from the column loading and washing phases consisted of pure cellulase, free of papain: the activity of the papain in the cellulase was below the limits of detection, based on the activity against azo-casein. The papain bound with S-Sepharose was subsequently recovered by passing 1.0 M sodium chloride, pH 4.8, over the resin. The volume of the Trichoderma cellulase preparation treated with protease was about 2,700 liters. The Trichoderma cellulase preparation treated with the resulting protease was preserved by adjusting its pH to 4.0 and adding sodium benzoate. This composition was then concentrated by ultrafiltration and conventionally stabilized ("Enzyme Applications", Encyclopedia of Chemical Technology, vol.9, fourth edition, 1994) at a final concentration of about 1,800 CMC units / ml.
Example 2 Denim Wash with a Trichoderma Cellulase Composition Treated with Protease A 35 Ib UniMac washer / extractor machine was used. Approximately 5.1 kg of discarded denim garments were placed in the machine. The denim consisted of 3 stitched pant legs, 30 cm long and 5 pieces of one square meter of 14 ounce Swift denim, No. 37628, and three 30-inch stitched pant legs of 12-ounce Swift denim , No. 25113, all made by Swift, of Drummondville, Quebec, Canada. The denim was dismantled by treatment for 15 minutes at 70 ° C with 30 g of the alpha-amylase enzyme Rapidase UC, available from Gist-Brocades. The machine was carried with 51 liters of hot water and brought to 50 ° C. The liquor ratio was 10: 1 (weight of liquor to weight of garments). The liquor was buffered to a pH of 6.0 with 300 g of 85% phosphoric acid and 114 g of sodium hydroxide beads. The machine was agitated for one minute to disperse the buffer and set the temperature. At this point, 70 ml of the protease treated cellulase preparation of Example 1 was added to the machine. The garments were washed at 47 rpm for 60 minutes. After the above, the bathroom was removed. The bath was then filled with water at 50 ° C and 2 g / l of caustic soda was added to adjust the pH to 9.0-11.0 to destroy the activity of the cellulase. The machine was shaken for 10 minutes and then the bath was removed. The garments were then rinsed with cold water for 5 minutes and hot water for 30 seconds. This was followed by two 10 minute rinses at 50 ° C, then the bathroom was removed and rotated. The garments were then dried in a conventional domestic dryer for 30 minutes. The garments were then removed from the dryer and ironed without steam. Denim brightness readings were taken using an Elrephro brightness meter. The pant leg brightness readings No. 37628 were used to estimate the net removal of dye and became the net amount of indigo dye removal from the fabric when compared with samples of known indigo content. The results are reported as a percentage of the indigo in unwashed denim. The brightness readings of the trouser leg No. 25113 were used to estimate the degree of retro-dyeing and were converted into the net amount of indigo dye re-deposited on the cloth by comparison with samples of known indigo content. The results are reported as a percentage of the indigo in unwashed denim. The procedure was repeated with 88 ml of cellulase logen, a cellulase enzyme that is re-deposited, conventional. The results are shown in Table 1. Table 1 Comparison of Trichoderma Cellulase Treated with Protease and Not Treated
As is evident from Table 1 for approximately the same degree of dye removal, the denim exposed to the protease-treated cellulase exhibits much less retro-dyeing than in the case of the cellulase that is re-deposited. Example 3 Washing of Denim with Surfactant Added Using the procedures described in Example 2, except as indicated, the following enzymes were used in denim washing: a. 80 ml of Trichoderma cellulase treated with protease of Example 1, used as in Example 2. b. 80 ml of Trichoderma cellulase treated with protease of Example 1, used as in Example 2, except that 40 ppm of Triton (registered trademark) X100 was added to the washing machine at the start of washing with cellulase. c. 80 ml of Trichoderma cellulase treated with protease of Example 1, used as in Example 2, except that 80 ppm of Triton (registered trademark) XlOO was added to the washing machine at the start of washing with cellulase. d. 80 ml of Trichoderma cellulase treated with protease of Example 1, used as in Example 2, except that 160 ppm of Triton (registered trademark) XlOO was added to the washing machine at the start of washing with cellulase. Table 2 Demonstration of the Effect of the Surfactant Added
As shown in Table 2, the addition of the surfactant further reduces the back-staining without significant loss of net release of colorant. Example 4 Comparative Results of Denim Washing Using the procedures described in Example 2, the following enzymes were used in the denim wash: a. Cellulase treated with protease of Example 1 and tested in Example 2. b. Following the protocols of Clarkson and collaborators, cellulase logen (which consists of 142 mg / ml of protein) was added as Trichoderma cellulase that is re-deposited and respectively 0.02, 0.10, 0.5, 2.5 and 12.5 ml of subtilisin Rapidase WSL-2, available from Gist-Brocades, were added as a protease. This protease consists of 110 mg / ml of protein. The amount of cellulase logen used was 88 ml, except for the run with 12.5 ml of protease, which had 78 ml of cellulase. The protease addition levels were respectively 0.02, 0.08, 040, 2.0 and 10% by weight of the subtilisin protein based on the weight of the cellulase protein. The minimum desirable level taught by Clarkson et al. Was 0.1%. As suggested by Clarkson et al., The pH in the washer was adjusted to 5.0 with 152 g of glacial acetic acid and 55 g of sodium hydroxide beads. All other procedures were as in Example 2. The results are reported in Table 3 and demonstrate that the cellulase composition of Trichoderma treated with protease gave a much lower level of back-staining than the method of Clarkson et al. Although the results contradict the teaching of Clarkson et al. On the need for protease in washing, support its teachings when cellulase and protease are added to the washing machine, the back-staining with more than 0.1% protease is considerably reduced to the cellulase present.
Table 3 Comparison of Cellulase Enzyme Compositions that Do Not Re-Deposit
Protease treated Approximately 1,000 liters of a natural Trichoderma cellulase preparation was produced by fermentation of Trichoderma longibrachiatum and dialyzed at a conductivity of 310 μS. Although this product was not stabilized nor conserved, it is available in a stabilized and conserved form as logen cellulase from logen Corporation. A substantially similar material can be prepared by simply diallylating logen cellulase to remove stabilizers and preservatives. It was then concentrated to a volume of 725 liters by ultrafiltration. The resulting dialysis product had a protein concentration of 98 g / l and an endoglucanase activity of 1.325 CMC units / ml using the method of Ghose (1987). 375 liters of the preparation were removed and the remaining preparation was then mixed with 150 liters of soft water and 15.75 kg of Folexco 300 MCU papain, available from Folexco Incorporated, and having an active papain protein concentration estimated at 32 g / kg and an estimated activity of 300 bed coagulant units (MCU) / mg of papain powder. The pH was adjusted to 4.8 using sodium benzoate and the mixture was incubated at approximately 35-50 ° C for 27 hours. The resulting treatment degree was approximately 24 g min / g. The inventors estimate that approximately 20% of the FPU activity and none of the CMCU activity were lost during this protease treatment. The mixture was then quenched at about 10 ° C for a period of two hours. The mixture was then rinsed over a silver pre-coated with diatomaceous earth and a frame filter. With rinse water, the preparation was diluted to a volume of approximately 800 liters. Protease was removed from the protease and cellulase mixture by passing the preparation over an S-Sepharose cation exchange resin according to the resin manufacturer's instructions (Pharmacia technical manual 18-1022-19"Ion Exchange Chromatography: Principles and Methods ", 1991). First S-Sepharose was equilibrated to a pH of 4.7 with acetate buffer; subsequently, the protease / cellulase mixture was loaded onto the resin to a degree of approximately 5 g of papain protein per liter of packed resin. The resin was washed with acetate buffer, pH 4.7. The combined effluents from the column loading and washing phases consisted of pure cellulase, free of papain: the papain activity in the cellulase was below the limits of detection based on activity against azo-casein. The papain bound with S-Sepharose was subsequently recovered by passing 1.0 M sodium chloride, pH 4.8, over the resin. The volume of the Trichoderma cellulase preparation treated with protease was about 1,600 liters. The Trichoderma cellulase preparation treated with the resulting protease was preserved by adjusting its pH to 4.0 and adding sodium benzoate. This composition was then concentrated by ultrafiltration and conventionally stabilized ("Enzyme Applications", Encyclopedia of Chemical Technology, vol.9, fourth edition, 1994) at a final concentration of approximately 2,000 CMC units / ml. Example 6: Washing of Denim with Trichoderma Cellulase Compositions Treated with Protease Using the procedures described in Example 2, except as noted, the following enzymes were used in the denim wash: a. 86 ml of Trichoderma cellulase treated with protease of Example 5, used as in Example 2. b. 70 ml of Trichoderma cellulase treated with protease of Example 1, used as in Example 2. c. 88 ml of cellulase logen, as described and tested in Example 2. The results are reported in Table 4 and demonstrate that the lower levels of papain treatment employed in Example 5 (24 g min / g) do not give as good operating as the most severe treatments of Example 1 (216 g min / g). Table 4 Comparison of Trichoderma Cellulase Treated with Protease and Not Treated
Example 7 Comparative Results of Denim Washing Using the procedures described in Example 2, the following enzymes were used in the denim wash: a. Cellulase treated with protease of Example 1 and tested in Example 2.
b. 80 ml of Trichoderma cellulase treated with protease of Example 1, tested as in Example 3 with 40 ppm of Triton XlOO added to the washing machine at the start of washing with cellulase. c. Euro L, a commercial cellulase product from Genecor
International that is believed to contain the protease enzyme, a Trichoderma cellulase that is re-deposited and a surfactant, was added in an amount of 100 ml. According to the manufacturer's suggestion, the pH was adjusted to 5.5 with 150 g of glacial acetic acid and 85 g of sodium hydroxide beads. All other procedures were as in Example 2. d. Euro L was added to the washer in an amount of 100 ml. 40 ppm of Triton XlOO was also added to the washing machine at the start of the cellulase wash. In accordance with the manufacturer's suggestion, the pH was adjusted to 5.5 with 150 g of glacial acetic acid and 85 g of sodium hydroxide beads. All other procedures were as in Example 2. e. Denimax L, a commercial product of Novo Nordisk that is believed to contain a low-dyeing enzyme made from Humicola insolens, was added in an amount of 250 ml. In accordance with the manufacturer's suggestion, the pH was adjusted to 6.5 with 297 g of phosphoric acid and 125 g of sodium hydroxide beads. All other procedures were as in Example 2. f. Denimax L was added to the washing machine in an amount of 250 ml. 44 ppm Triton XLOO was also added to the washer at the start of the cellulase wash. In accordance with the manufacturer's suggestion, the pH was adjusted to 6.5 with 297 g of phosphoric acid and 125 g of sodium hydroxide beads. All other procedures were as in Example 2. The results are reported in Table 5 and demonstrate that, for similar release of dye, the Trichoderma cellulase composition treated with protease gives the denim a lower level of retro-dyeing than Euro L or Denimax L. This good performance of Trichoderma cellulase treated with protease without surfactant, in relation to Euro L, is achieved even when Euro L contains performance enhancing surfactants. The low potency of the Humicola insolens cellulase is evident from the fact that it took two to three times more Denimax L to fade the denim than the case of other enzymes. Table 5 Comparison of Cellulase Enzyme Compositions That Are Not Replaced
Although preferred embodiments of the invention have been shown and described, the invention should be defined solely by the scope of the appended claims.