# US 5,700,686 A · Protease-treated and purified cellulase compositions and methods for reducing backstaining during enzymatic stonewashing > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,700,686 A - Document ID: US5700686A - Status: Granted - Kind: A - Application number: 08/466,424 - Filed: 1995-06-06 - Granted: 1997-12-23 - Earliest claimed: 1995-06-06 - Assignees: Iogen Corporation - Inventors: Brian Foody; Colin Nicholson; Jeffrey Tolan; Theresa White - Web page: https://patentgrep.com/patent/US5700686A ## CPC - C12N 9/2437: {Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)} - C11D 3/38645: {containing cellulase} - C12Y 302/01004: Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase - C12Y 302/01091: Cellulose 1,4-beta-cellobiosidase (3.2.1.91) - D06P 5/158: {with other compounds} - Y10S 435/945: Trichoderma - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL - C12Y: ENZYMES - D06P: DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS ## Abstract During the enzymatic "stone washing" of a denim fabric and/or garments, an undesirable redeposition of blue dye often occurs on the surfaces of the denim. The invention relates to a means of overcoming this problem using an enzyme composition comprised of Trichoderma endoglucanase and Trichoderma cellobiohydrolase that has been partially digested by a protease enzyme to separate its core and binding domains. The use of this composition reduces the redeposition of the blue dye and hence improves the stone washing process relative to using a redepositing or backstaining cellulase. ## Claims ### Claim 1 A cellulase enzyme composition that produces low backstaining of indigo-dyed denim relative to a natural Trichoderma cellulase, the enzyme composition comprising a protease-treated Trichoderma cellulase that is a product of a process comprising: a. subjecting a Trichoderma cellulase containing a cellobiohydrolase enzyme and an endoglucanase enzyme to a limited protease treatment by adding a protease to the cellulase, the limited protease treatment being defined by using a weight ratio of protease to cellulase multiplied by an average treatment time that is substantially between 1.0 and 10,000 gram minutes per gram, b. stopping the protease reaction by chilling or adjusting the pH, and c purifying the cellulase by substantially removing the added protease so as to define the composition. ### Claim 2 (depends on 1) The composition composition according to claim 1, wherein said protease treats the cellulase to an extent that the treated cellulase loses at least 5% of its initial activity as measured in filter paper units (FPUs) and no more than 50% of initial activity as measured in carboxymethylcellulose CMC units. ### Claim 3 (depends on 1) The composition according to claim 1 wherein the protease treats the cellulase to an extent that the treated cellulase maintains substantially between 15% and 90% of its initial activity as measured in FPUs, and maintains substantially between 70% and 100% of its initial activity as measured in CMC units. ### Claim 4 (depends on 1) The composition according to claim 1 wherein the extent of said limited protease treatment step is defined by using a weight ratio of protease to cellulase multiplied by an average treatment time that is substantially between 10.0 and 5,000 g min/g. ### Claim 5 (depends on 1) The composition according to claim 1 wherein the added protease comprises a papain protease. ### Claim 6 (depends on 1) The composition according to claim 1 wherein the added protease remaining the composition after the purification step is less than 0.1%, by weight of the composition, as measured by total protein content. ### Claim 7 (depends on 1) The composition according to claim 1 wherein the added protease remaining in the composition after the purification step is less than 0.05%, by weight of the composition, as measured by total protein. ### Claim 8 (depends on 1) The composition according to claim 1 wherein no detectable added protease remains in the composition after the purification step. ### Claim 9 (depends on 1) A composition according to claim 1, wherein the Trichoderma cellulase to be treated further comprises endoglucanase (EG) enzyme and cellobiohydrolase (CBH) core domains, wherein at least some of the CBH core domains are not linked to their natural binding domains and wherein the composition defined has substantially no incremental or residual protease activity over the amount which is produced naturally by the Trichoderma. ### Claim 10 (depends on 9) A composition according to claim 9, wherein said cellulase to be treated further is made by a strain of Trichoderma that was genetically modified to produce directly at least some of the CBH core domains which are not linked to their natural binding domains. ### Claim 11 A cellulase enzyme composition that produces low backstaining of indigo-dyed denim relative to a natural Trichoderma cellulase, the enzyme composition comprising a protease-treated Trichoderma cellulase that is a product of a process comprising: (a) subjecting a Trichoderma cellulase containing a cellobiohydrolase enzyme and an endoglucanase enzyme to a limited protease treatment by adding a protease to the cellulase, the limited protease treatment being defined by using a weight ratio of protease to cellulase multiplied by an average treatment time that is substantially between 1.0 and 10,000 gram minutes per gram, and immediately, (b) purifying the cellulase by substantially removing the added protease so as to define the composition. ### Claim 12 (depends on 11) The composition according to claim 11, wherein said protease treats the cellulase to an extent that the treated cellulase loses at least 5% of its initial activity as measured in filter paper units and no more than 50% of its initial activity as measured in CMC units. ### Claim 13 (depends on 11) The composition according to claim 11 wherein the protease treats the cellulase to an extent that the treated cellulase maintains substantially between 15% and 90% of its initial activity as measured in FPUs, and maintains substantially between 70% and 100% of its initial activity as measured in CMC units. ### Claim 14 (depends on 11) The composition according to claim 11 wherein the extent of said limited protease treatment step is defined by using a weight ratio of protease to cellulase multiplied by an average treatment time that is substantially between 10.0 and 5,000 g min/g. ### Claim 15 (depends on 11) The composition according to claim 11 wherein the added protease comprises a papain protease. ### Claim 16 (depends on 11) The composition according to claim 11 wherein the added protease remaining in the composition after the purification step is less than 0.1% by weight of the composition, as measured by total protein content. ### Claim 17 (depends on 11) The composition according to claim 11 wherein the added protease remaining in the composition after the purification step is less than 0.05%, by weight of the composition, as measured by total protein. ### Claim 18 (depends on 11) The composition according to claim 11 wherein no detectable added protease remains in the composition after the purification step. ### Claim 19 A cellulase enzyme composition that produces low backstaining of indigo-dyed denim relative to a natural Trichoderma cellulase, the enzyme composition comprising a protease-treated Trichoderma cellulase that is a product of a process comprising: (a) subjecting a Trichoderma cellulase containing a cellobiohydrolase enzyme and an endoglucanase enzyme to a limited protease treatment by adding a protease to the cellulase, the limited protease treatment being defined by using a weight ratio of protease to cellulase multiplied by an average treatment time that is substantially between 1.0 and 10,000 gram minutes per gram, and (b) stopping the protease reaction by chilling or adjusting the pH. ### Claim 20 (depends on 19) The composition according to claim 19, wherein said protease treats the cellulase to an extent that the treated cellulase loses at least 5% of its initial activity as measured in filter paper units and no more than 50% of its initial activity as measured in CMC units. ### Claim 21 (depends on 19) The composition according to claim 19, wherein the protease treats the cellulase to an extent that the treated cellulase maintains substantially between 15% and 90% of its initial activity as measured in FPUs, and maintains substantially between 70% and 100% of its initial activity as measured in CMC units. ### Claim 22 (depends on 19) The composition according to claim 19, wherein the extent of said limited protease treatment step is defined by using a weight ratio of protease to cellulase multiplied by an average treatment time that is substantially between 10.0 and 5,000 g min/g. ### Claim 23 (depends on 19) The composition according to claim 19, wherein the added protease comprises a papain protease.