WO1997011165A9 - Protease modified cellulase compositions - Google Patents

Abstract

A modified cellulase and method of preparing is disclosed which comprises a cellulase composition which is treated with a protease wherein at least one proteolytically stable cellulase is present in the cellulase composition. The disclosed cellulase composition may be used in the treatment of lignocellulosic material, the treatment of animal feed, the treatment of waste water, the treatment of textiles, stonewashing or in laundry detergents.

Description

PROTEASE MODIFIED CELLULASE COMPOSITIONS

BACKGROUND OF THE INVENTION

The present invention is related to a modified cellulase composition which results from treating cellulase with protease In particular, the present invention is related to a modified cellulase composition in which specific components of the cellulase are differentially stable to proteolysis such that protease treatment preferentially degrades certain protein and cellulase components while leaving other cellulase and protein components relatively intact The present invention contemplates use of the protease treated cellulase composition in textile treatment, pulp and paper production, gram and animal feed processing and detergents

Cellulases are enzymes which break down cellulose by hydrolyzing the β-1 ,4-glucan linkages to form glucose, cellobiose and various cello-oligosacchaπdes Cellulases expressed in certain fungi and bacteria have been extensively characterized For example cellulase produced by the fungal species Tπchoderma sp (especially Tnchoderma longibrachiatum), has been subject to a high level of attention due to the capability of the species to produce in large quantities a complete cellulase system ("whole cellulase") which is effective in degrading crystalline forms of cellulose Similarly, cellulases produced by fungal species such as Neurospora sp , Peniαllium sp , Fusanum sp , Humicola sp and Aspergillus sp and bacterial cellulases, for example those derived from Bacillus have also been the subject of much study

Complete fungal cellulase systems comprise several different enzyme classifications including those identified as exo-cellobiohydrolases (EC 3 2 1 91) ("CBH") endoglucanases (EC 3 2 1 4) ("EG"), and β-glucosidases (EC 3 2 1 21) ("BG")(see e g Schulein, "Methods in Enzymology", 160, 25, pages 234 et seq (1988)) Each of the fungal cellulase classifications of CBH, EG and BG can be further expanded to include multiple components within each classification For example, cellulase compositions containing multiple CBHs, EGs and BGs have been isolated from a variety of fungal sources

The complete cellulase system comprising CBH, EG and BG components is required to efficiently convert crystalline cellulose to glucose Isolated components are far less effective, if at all, in hydrolyzing crystalline cellulose to glucose Moreover, a synergistic relationship is observed between the cellulase components particularly if they are of different classifications For example, combinations of specific EG components and specific CBH components may be more or less efficient in breaking down cellulose Similarly, vaπous cellulase components or subcombinations thereof have been found to be more useful in industrial applications than corresponding complete cellulase systems. For example, problems have been observed when using certain cellulase components, e.g., CBH I, CBHII, EGI and EGII components derived from Trichoderma longibrachiatum, in detergents and textile treatment compositions due to fabric strength loss and dye redeposition, also known as backstaining.

U.S. Patent No. 5,120,463 discloses that CBHI enriched cellulase compositions provide excellent cleaning characteristics while reducing fiber degradation effects.

U.S. Patent No. 5,246,853 discloses that the presence of exo-cellobiohydrolase type components in combination with endoglucanase type components in a stonewashing compositions results in detrimental strength loss.

U.S. Patent No. 5,290,474 discloses that the EGlll component of Trichoderma spp. provides for superior and unexpected advantages in detergent compositions as compared to the EGI and EGII components of Trichoderma reesei and that certain exo- cellobiohydrolase type components are responsible for undesirable characteristics such as increased strength loss.

The various components of complete cellulase systems are also known to have differing optimal pH and pH activity profiles which can both adversely and advantageously effect their industrial application. For example, while isolated endoglucanase type components are of value in the textile industry due to the desirable characteristics they confer to fabrics, many fungal endoglucanase components have their maximal activity at acidic pHs whereas most laundry detergent compositions are formulated for use at from neutral to alkaline (pH >7 to about 10) conditions. EGlll derived from Trichoderma longibrachiatum is known to have a relatively neutral pH activity profile and significant alkaline pH activity. In addition to its use in laundry detergents, substantially pure EGlll cellulase component can be used in a pre-washing step at an intermediate pH where sufficient activity exists to provide desired improvements in color retention and restoration, softening and feel as disclosed in U.S. Patent No. 5,290,474. EGlll cellulase component has also been described for home use in a stand alone composition suitable for restoring color to faded fabrics, see e.g., U.S. Patent No. 4,738,682. The high activity under neutral to alkaline conditions of EGlll is also beneficial in textile processes for treating cotton- containing fabrics (see U.S. Serial Nos. 07/954,113 and 08/210) as well as in silage and/or composting processes.

However, EGlll represents only between 1-4% of the total cellulase protein expressed in Trichoderma longibrachiatum making purification from other cellulases essential to obtain significant EGlll activity. U.S. Patent No. 5,328,841 discloses a method for punfying EGlll and xylanase from an extracellular culture media including a mixture of cellulase and xylanase by adding polyethylene glycol having a molecular weight of between about 5,000 and 10,000 Genetic engineering techniques have also been used to ease preparation of specific cellulase components from whole cellulase For example, PCT Publication No WO 92/06209 discloses preparation and cultivation of Tnchoderma reesei strains which are deleted for one or more cellulase genes, thus producing enriched concentrations of, e g , EGI, EGII, EGlll, CBHI or CBHII compared to the complete cellulase systems

Fermentation of cellulase producing microorganisms, however, in addition to presenting the problem of expression of undesirable cellulase components, also result in significant background protein production For example, a typical fermentation broth may include peptide compounds which deleteriously effect certain cellulase uses These peptide components are believed to cause adverse effects in cellulase applications, for example, backstaining in stonewashing processes

Thus, the pπor art illustrates the significant uses for specific cellulase components However, a continuing need exists in the pnor art for simple and effective methods of modifying cellulase compositions to eliminate specific components and background protein while retaining certain desired components

SUMMARY OF THE INVENTION

It is an object of the present invention to provide for a simple and effective way to increase the relative activity of desired cellulase components when compared to less desirable cellulases which are present in complete cellulase systems

It is a further object of the present invention to provide for a simple and effective way of removing background proteins which carry over from fermentation processes and which deleteriously effect certain cellulase uses by causing, e g , backstaining

The present invention is directed to the discovery that certain cellulase components and other peptides are differentially degraded by protease More importantly, however, the present invention is directed to the discovery that cellulase modified according to the present invention will have desirable properties over unmodified cellulase This discovery allows the use of protease modification of cellulase to produce a composition which has desirable attributes, e g , decreased backstaining and/or decreased strength loss in textile applications The present invention achieves the above stated objects through the use of a cellulase composition which has been modified with protease either simultaneously with or prior to, the use of the cellulase composition in an intended application According to the present invention, a method of preparing a modified cellulase is provided compπsing the steps of (a) adding protease to a cellulase composition comprising at least one proteolytically stable cellulase; and (b) incubating said cellulase and said protease in an aqueous solution for a time and under conditions sufficient to degrade non- proteolytically stable cellulases.

According to a composition embodiment of the invention, a modified cellulase composition is prepared according the above method Preferably, the modified cellulase composition so prepared is present in a detergent composition or a stonewashing composition for the treatment of textiles

The invention itself, together with further objects and attendant advantages, will best be understood by reference to the following detailed description taken in conjunction with the accompanying drawings

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows an isoelectric focusing gel of an enzyme mixture after protease treatment on specific components of Tnchoderma longibrachiatum whole cellulase

DETAILED DESCRIPTION OF THE INVENTION

"Cellulase proteins" refers to enzymes including exo-cellobiohydrolase (CBH) proteins, endoglucanase (EG) proteins and β-glucosidase (BG) proteins derived from wild- type fungal sources or microorganisms genetically modified so as to incorporate and express all or part of the cellulase genes obtained from a wild-type fungal sources Collectively, all of such proteins (i e , CBH, EG and BG proteins) are referred to as "cellulase proteins". Contranly, cellulase proteins do not include other proteins expressed by cellulase producing organisms such as, for example xylanases, proteases, amylases

"Complete cellulase" or "whole cellulase" refers to a composition comprising a cellulase profile representative of that produced by a naturally occurring cellulase producing microorganism For example, Tnchoderma longibrachiatum whole cellulase prepared under standard fermentation conditions will show a cellulase profile which contains approximately the following amount of the following components 45-55% exo-cellobiohydrolase I ("CBHI ), 13-15% exo-cellobiohydrolase II ("CBHII"), 11-13% endoglucanase I ("EGI"), 8- 10% endoglucanase II ("EGII"), 1-4% endoglucanase III ("EGlll") and 0 5-1 % β-glucanase ("BG"), by weight of cellulase

"Modified cellulase" refers to a cellulase composition, for example a whole cellulase or portion thereof, which has been treated with protease to modify the relative activities therein Thus, where complete cellulase from Tnchoderma longibrachiatum is modified the resulting modified cellulase will comprise a relative increase in activity of EGlll and relative decrease in the activity of CBHI, CBHII, EGI and EGII compared to the complete cellulase

"Cellulose binding domain" or "binding domain" refers to a distinct structural attribute of many cellulases which is responsible for attaching a cellulase to cellulose "Binding domain" as referred to herein means the structure known to exist in many cellulases comprised of a distinct sequence of ammo acids which is attached via a linker region to a catalytic core subunit "Cellulose binding domain" does not refer to a "binding region" which is structurally integral to the catalytic core

"Endoglucanase" or "EG" refers generally to a cellulase enzyme which hydrolyzes the internal β(1-4) glycosidic linkages of a cellulose polymer Endoglucanases are generally characterized by their ability to (a) hydrolyze soluble cellulose derivatives such as carboxymethylcellulose ("CMC"), thereby reducing the viscosity of CMC containing solutions, and (b)readιly hydrolyze hydrated forms of cellulose such as phosphoric acid swollen cellulose (e g , Walseth cellulose) and hydrolyze less readily the more highly crystalline forms of cellulose (e g , Avicel, Solkafloc, etc ) Microbial sources often contain more than one type of endoglucanase The different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificities and different enzymatic action patterns For example, EGI, EGII and EGlll from Tnchoderma longibrachiatum are all characterized by different biochemical characteristics

"EGlll cellulase" refers to the endoglucanase component derived from Tnchoderma spp or any microorganism producing a protein equivalent to EGlll produced by Tnchoderma spp characterized by a pH optimum of about 5 5 to 6 0, an isoelectric point (pl) of from about 7 2 to 8 0, and a molecular weight of about 23 to 28 Kdaltons Preferably, EGlll cellulase is derived from either Tnchoderma longibrachiatum or from Tnchoderma vmde EGlll cellulase denved from Tnchoderma longibrachiatum has a pH optimum of about 5 5 to 6 0, an isoelectric point (pl) of about 7 4 and a molecular weight of about 25 to 28 Kdaltons EGlll cellulase derived from Tnchoderma vmde has a pH optimum of about 5 5, an isoelectric point (pl) of about 7 7 and a molecular weight of about 23 5 Kdaltons Additionally, it is contemplated that the ammo acid sequence of the EGlll cellulase may be altered Alteration of the active sites on this enzyme may lead to a variety of changes such as altered pH optima, altered temperature optima or altered affinities for the substrate EGII has been previously referred to by the nomenclature "EGlll" by some authors but current nomenclature uses the term "EGII" The EGII protein is recognized to be substantially different from the EGlll protein in its molecular weight, pl, and pH optimum as evidenced by Table 2 of Example 2 presented below.

"Exo-cellobiohydrolase" or "CBH" refers to a cellulase enzyme properly known as cellulose β(1-4)cellobiosidase. CBH components are generally believed to cleave glucose and cellobiose units from the end of the cellulose polymer. CBH components are generally characterized by the fact that (a) they are competitively inhibited by cellobiose (K, approximately 1mM); (b) they are unable to significantly hydrolyze substituted celluloses such as carboxymethylcellulose and (c) they hydrolyze phosphoric acid swollen cellulase and to a lesser degree highly crystalline cellulose. Similar to the EG's, a single microbial source can produce a variety of CBH components. For example, Trichoderma longibrachiatum is known to produce at least two distinct CBH components, CBHI and CBHII.

"β-Glucosidase (BG) components" refer to those components of cellulase which exhibit BG activity; that is to say that such components will act from the non-reducing end of cellobiose and other soluble cellooligosaccharides ("cellobiose") and give glucose as the sole product. BG components do not adsorb onto or react with cellulose polymers. Furthermore, such BG components are competitively inhibited by glucose (K, approximately 1mM). While in a strict sense, BG components are not literally cellulases because they cannot degrade cellulose, such BG components are included within the definition of the cellulase system because these enzymes facilitate the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly cellobiose) produced by the combined action of CBH components and EG components. Without the presence of BG components, moderate or little hydrolysis of crystalline cellulose will occur BG components are often characterized on aryl substrates such as p-nitrophenol B-D- glucoside (PNPG) and thus are often called aryl-glucosidases. It should be noted that not all aryl-glucosidases are BG components, in that some do not hydrolyze cellobiose.

The various cellulase components are further characterized in the characteristics they confer to products which incorporate them. For example, stonewashing compositions are known to be adversely effected by CBH components which cause an increased level of backstaining. This backstaining results in a less desirable finished textile product. Further, the presence of both EG components and CBH components in any textile processing composition will result in excessive strength loss in the treated textiles. Thus, both EG and CBH components have been proposed for use in detergents alone. Accordingly, methods, such as the present invention, to selectively remove one or the other of these components are desirable. "Proteolytically stable cellulase" refers to a cellulase which is differentially resistant to proteolytic attack compared to other cellulases in the same incubation mixture. For example, complete cellulase derived from Trichoderma longibrachiatum contains a component mixture of at least the following cellulases: CBHI, CBHII, EGI, EGII and EGlll. However, when subjected to protease treatment, only the EGlll component retains significant activity. Components which behave in a manner similar to EGlll will confer similar results and are contemplated as within the invention. Accordingly, cellulases having a level of homology of amino acid sequence to the amino acid sequence of EGlll from T. longibrachiatum of greater than 60%, preferably greater than 70% and most preferably greater than 90% will confer similar benefits in a protease/cellulase solution wherein it is desired to retain only significant activity from one such component (see PCT Publication No. WO 94/21801). Similarly, Applicants believe that cellulases lacking binding domains, such as EGlll from T. longibrachiatum and many cellulases derived from bacterial species such as Bacillus, will possess similar proteolytic resistance and will be valuable in protease/cellulase compositions.

Accordingly, in a modified cellulase according to the present invention, the composition will retain a significant amount of activity corresponding to the proteolytically stable cellulase while the activity of non-proteolytically stable cellulases will be significantly diminished. Preferably, the remaining activity after protease treatment for the proteolytically stable cellulase will be greater than about 10 percent, more preferably greater than about 15 percent; and most preferably greater than about 30 percent relative to unmodified proteolytically stable cellulase. Similarly, the remaining activity after protease treatment of non-proteolytically stable cellulase will preferably be less than about 10 percent relative to unmodified non-proteolytically stable cellulase.

In a preferred embodiment of the invention, a cellulase mixture containing a desired proteolytically stable cellulase is incubated with protease for an appropriate time and under appropriate conditions to substantially hydrolyze or degrade non-proteolytically stable cellulases and background proteins in the solution. The resulting modified cellulase solution is then optionally subjected to well known separation techniques to remove the protease and/or the hydrolyzed contaminating proteins. In such a way, it is possible to reduce or eliminate the activity of certain cellulases while increasing the activity of proteolytically stable cellulases. In a further embodiment, it is possible to remove the hydrolyzed proteins and enzymes which results in a substantially purified cellulase mixture in proteolytically stable cellulase. The cellulase composition useful in the present invention may be derived from any source, e g , a fungal or bacterial source Preferred cellulases which may contain mixtures of proteolytically stable cellulases and non-proteolytically stable cellulases include those derived from fungal sources such as Tnchoderma, Humicola, Aspergillus, Streptomyces, Erwinia, Neurospora, Penicillium, Thermomonospora, Microtetraspora or Fusanum or from bacterial sources such as Bacillus Particularly preferred are the cellulases of Tnchoderma longibrachiatum Methods for preparing cellulase solutions from sources such as those listed above are well known in the art The cellulase mixture according to the present invention may be either an aqueous solution or a dry solid, e g , a granular composition comprising cellulase

The protease according to the invention may be derived from either exogenous or endogenous sources Added exogenous protease refers to proteases that differ from those naturally present in the enzyme complement of the microorganism from which the cellulase composition to be treated is derived Alternatively, the added protease may be endogenous In this case, added endogenous proteases refers to an amount of protease in a redepositing cellulase composition that is over and above what is naturally produced by the microorganism or produced via overexpression of the gene encoding the naturally occurring protease Proteases are available from several sources including microbial, plant and animal sources Preferred proteases are derived from Bacillus licheniformis, Bacillus subtilis and Aspergillus oryzae Proteases suitable for the invention include, for example, seπne, metallo and acid proteases, as well as endo- and exo-proteases Subtihsins are serine proteases which generally act to cleave internal peptide bonds of proteins and peptides Metallo proteases are exo- or endo-proteases which require a metal cofactor for activity One of the preferred serine proteases is subtilism Particularly preferred proteases useful in the present invention are proteases obtained from a microorganism genetically modified as descπbed in US Patents Nos 5,182,204, 5,185,258 and 5,316,941 and U S Patent No Re 34,606 the disclosures of which are incorporated herein by reference

The amount of added protease is determined by many factors such as the purity, concentration, activity and specificity of the added protease, as well as the length and temperature of the incubation with the cellulase composition Therefore, the amount of added protease will vary with the type of protease added to the cellulase composition

Where the protease added is derived from the same source as the cellulase and the cellulase is complete cellulase, the amount of protease added according to the present invention is an amount of protease in excess of the amount which is produced naturally by a microorganism The added protease will proteolytically cleave the contaminating proteins, thus selectively retaining the desired proteolytically stable cellulase

Conditions appropriate to hydrolyze the contaminating proteins upon incubation of the cellulase and protease will be dependent on the type of protease used. Generally, conditions sufficient for proteolytic activity of a specific protease used will be suitable for the present purposes Of course, the closer the incubation conditions are to the optimal conditions for the protease, the more efficient the hydrolysis of contaminating proteins will be. The temperature of the incubation should preferably be between 20 and 70°C, more preferably between 30 and 35°C The time of incubation should preferably be shorter than 24 hours, more preferably between 1 and 120 minutes, and most preferably between 10 and 60 minutes The pH of the incubation is preferably between about 4 and 10, more preferably between about 6 and about 8, depending on the pH optimum of the protease However, the pH should not be so high as to oxidize or denature the proteolytically stable cellulase

Subsequent to modification, the protease or hydrolyzed proteins may be optionally removed from the solution Removal of the protease or hydrolyzed proteins from the proteolytically stable cellulase may be achieved through any art-recognized means For example, the protease may be separated by precipitation, centrifugation, ultrafiltration, gel filtration, membrane filtration, ultramembrane filtration, affinity chromatography, pH or temperature inactivation Similarly, the hydrolyzed cellulases and background proteins may be removed from solution according to such art-recognized means Alternatively, the hydrolyzed proteins may be removed from the solution in conjunction with standard washing procedures For example, when the protease/cellulase composition is used in a detergent to clean dirty fabrics, the protease/cellulase composition will generally be added during either the pre-wash or regular wash cycles Upon being added to solution, the protease will degrade the background proteins and preferentially degrade non- proteolytically stable cellulases leaving a substantial portion of the proteolytically stable cellulases in solution As the proteolytically stable cellulases will tend to adhere to the fabrics to a greater degree than the degraded protein, upon removal of the wash liquor from the laundry chamber and replacement with either a subsequent wash cycle liquor or a rinse liquor, the degraded cellulases and background proteins will be removed leaving the intact cellulase in solution without the presence of the degraded background proteins and non-proteolytically stable cellulases.

The composition according to the present invention may be used for any purpose in which cellulase finds application For example, the present invention contemplates the use of the compositions disclosed herein in detergents, stonewashing applications, animal feed additives, and the treatment of lignocellulosic material and waste water U S Patent No 5,290,474, which disclosure is incoφorated by reference, discloses how to make and use detergent compositions comprising EGlll Similarly, U S Patent No 5,246,853, which disclosure is herein incoφorated by reference, discloses how to make and use compositions for treating cotton containing textiles, e g , stonewashing, utilizing EGlll

The present invention provides a significant advance in the preparation of cellulase components As illustrated in the example below, protease treatment of complete cellulase from Tnchoderma longibrachiatum preferentially hydrolyzes proteins and cellulases other than EGlll Thus, cellulases which are known to cause problems in industrial applications for example CBH-type cellulases, are eliminated from solution leaving a relatively purified EGlll sample in terms of remaining activity Similarly, background proteins are hydrolyzed preferentially to EGlll Applicants believe that these background proteins are responsible for numerous undesirable effects in industrial enzyme mixtures For example, background proteins may result in allergenicity which is inappropriate for specific uses, e g , laundry detergents Additionally, background proteins may cause poor results in applications in which the cellulase is used, for example, in stonewashing where they may cause backstaining

EXAMPLE

The protease sensitivity of cellulase components derived from Tnchoderma reesei was analyzed Whole cellulase and purified EGI, EGII, EGlll, CBHI and CBHII (as described in U S Patent No 5,246,853) were buffer exchanged into 20 mM TES pH 7 5 using 5 ml sizing columns (Pharmacia, PD10 columns, catalog # 17-0851-01 ) Each enzyme solution was diluted in 20 mM TES pH 7 5 buffer and separated into two 0 5 ml aliquots A concentrated subtilism protease solution derived from Bacillus subtilis was added to one aliquot, to give a final protease concentration of 0 4 mg/ml, and cellulase concentration of 4 mg/ml The same volume of buffer was added to the other enzyme aliquot

These samples were then placed at 37 ' C and incubated without shaking for 14 hours The samples were analyzed by first running them on an isoelectric focusing gel (Crescent Chemicals Servalyt precotes IEF gel, 3-10, catalog # 42965) which was overlaid for CMC activity The samples containing endoglucanases were assayed for endoglucanase activity using RBB-CMC (Megazyme, AZO-CMCellulose) as the substrate according to the method described in U S Patent No 5 246,853 As shown in Figure 1 , protease treatment leads to dramatically altered cellulase profiles for certain cellulases, e.g., EGI, EGII, CBHI and CBHII. Table 1 illustrates the results of activity testing of endoglucanase samples treated as above with protease.

TABLE 1

ENZYME NO PROTEASE ADDED PROTEASE ADDED

Whole cellulase from T. 100% Activity 8% Activity reesei

EGI 100% Activity < 2% Activity

EGII 100% Activity 3% Activity

EGlll 100% Activity 37% Activity

As shown in Table 1 , when EGlll is treated with protease, significantly more activity remains than when EGI, EGII or whole cellulase derived from T. reesei are treated with protease.

Of course, it should be understood that a wide range of changes and modifications can be made to the preferred embodiments described above. It is therefore intended to be understood that it is the following claims, including all equivalents, which define the scope of the invention.

Claims

We claim

1 A method of preparing a modified cellulase comprising the steps of

(a) adding protease to a cellulase composition comprising at least one proteolytically stable cellulase,

(b) incubating said cellulase and said protease in an aqueous solution for a time and under conditions sufficient to degrade non-proteotytically stable cellulases

2 The method according to claim 1, comprising the additional step of removing said protease from said solution after step (b)

3 The method according to claim 1 , comprising the additional step of removing hydrolyzed proteins from solution after step (b)

4 The method according to claim 1 wherein said proteolytically stable cellulase lacks a binding domain

5 The method according to claim 4, wherein said proteolytically stable cellulase has at least 60% homology to Tnchoderma longibrachiatum EGlll

6 The method according to claim 1 , wherein said protease is derived from Bacillus

7 The method according to claim 1 , wherein said protease is derived from Bacillus licheniformis and comprises a methionine at position 222

8 A composition of matter compπsing a modified cellulase produced by a process according to claim 1

9 The composition of matter according to claim 8, wherein said proteolytically stable cellulase has at least 60% homology to Tnchoderma longibrachiatum EGlll

10 The composition of matter according to claim 8, wherein said proteolytically stable cellulase lacks a cellulose binding domain

11 The composition of matter according to claim 8, wherein said proteolytically stable cellulase is EGlll

12 A detergent comprising the composition according to claim 8

13 A detergent comprising the composition according to claim 9

14 A detergent comprising the composition according to claim 10

15 A stonewashing composition comprising the composition according to claim 8

16 A stonewashing composition comprising the composition according to claim 17 A stonewashing composition comprising the composition according to claim 10

18 The use of the composition according to claim 8 in the treatment of lignocellulosic material

19 The use of the composition according to claim 8 in the treatment of waste water

20 The use of the composition according to claim 8 as an animal feed additive

21 The use of the composition according to claim 8 for treating textiles

22 The use of protease to selectively degrade cellulase which lack binding domain and other proteins over cellulase which possess a binding domain

23 The use according to claim 22 wherein said proteins which are selectively degraded includes protein which cause backstaining

24 The use of the composition according to claim 8 in a detergent