Classifications

• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
  • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
  • D06P5/15—Locally discharging the dyes
  • D06P5/158—Locally discharging the dyes with other compounds
• • C—CHEMISTRY; METALLURGY
  • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
  • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
  • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
  • C11D3/16—Organic compounds
  • C11D3/38—Products with no well-defined composition, e.g. natural products
  • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
  • C11D3/38645—Preparations containing enzymes, e.g. protease or amylase containing cellulase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
  • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
  • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06P—DYEING OR PRINTING TEXTILES; DYEING LEATHER, FURS OR SOLID MACROMOLECULAR SUBSTANCES IN ANY FORM
  • D06P5/00—Other features in dyeing or printing textiles, or dyeing leather, furs, or solid macromolecular substances in any form
  • D06P5/02—After-treatment

Definitions

• the present invention is directed to improved cellulase composition for treatment and finishing of cellulose-containing textile materials without causing significant strength loss to the materials.
• the cellulase composition of the present invention can be used to improve the appearance, softness, drapability, absorption of moisture, and dyeability of cellulose-containing textile materials, and to reduce the tendency of pilling and fuzzing. Additionally, the cellulase composition of this invention can be used in the finishing of so-called denims to create a so-called stone-washed appearance.
• Biofinishing has been used e.g. to remove all kinds of impurities and individual loose fibre ends that protrude from the textile surface.
• the key benefits offered by biofinishing with cellulases are permanent improval of depilling, cleared surface structure by reduced fuzz, improved textile handle, such as softness, smoothness and a silkier feel, improved drapability and dyeability of the textile and improved moisture absorbability.
• cellulases have been used to impart a stone-washed appearance to denims. Complete biodegradation of cellulases is an advantage of cellulase treatment, which consequently stands out as an environmentally-friendly alternative for chemical treatment.
• CYTOLASE 123 -cellulase (Genencor Int.) was used with no detailed composition given in the said publication.
• the cellulase solution may contain buffers, surfactants, abrasion agents, and the like. After treatment, the tensile strength of the cotton woven fabric was reported to be at least 50 percent of the tensile strength of untreated fabric.
• cellulase derived from fungi for example from Trichoderma reesei
• cellulase being composed of cellobiohydrolase (CBH), endoglucanase (EG) and beta-glucosidase (BG) type components.
• CBH and EG components can further be divided into CBHI and CBHII types and into several various EG types, the main types of the latter being EGI and EGII.
• the BG components do not react with cellulosic polymers, but further cleave the degradation products, for example cellobiose, that are formed as a result of the synergistic effect of the CBH and EG components.
• the original cellulase composition derived from microbes, for example from 7. reesei fungi, seldom is suitable to give a desired result.
• the original cellulase composition may comprise about 45-80% CBHI, 10-25% CBHII, 5-15% EGI and 8-15% EGII of the total cellulase protein content.
• the interrelations of the CBH and EG components contained in a composition may hence be changed by various methods known to persons skilled in the art. Such methods include, for example, fractionation and genetic engineering.
• 5,120,463 discloses a detergent composition
• a detergent composition comprising a surfactant and additionally 0.002 to 10 weight percent of cellulase composed of CBHI type and EG type components with the ratio of CBHI to (EGI + EGII) being > 10 : 1.
• the patent application PCT/US93/04149 discloses a method for treating cotton fabrics with fungal cellulase compositions comprising CBHI type and EG type components in a weight ratio greater than 10 : 1.
• Cellulases have been employed also in the finishing of denim fabrics or denim garments, in order to impart a stone-washed appearance to the fabric.
• pumice stones The stone-wash was traditionally performed using so-called pumice stones.
• pumice stones causes laundries several problems, such as the heaviness of handling the stones, the laborious picking by hand of the stones from among the garments, significant wear to the machines with resulting high repair and investment costs, the growing amounts of waste caused by broken stones and, additionally, the complicated access to pumice stones, as the mining of pumice stones is forbidden in certain countries on environmental grounds.
• a method for imparting a stone-washed appearance to denim garments by cellulase enzymes is disclosed, for example, in the US Patent No. 4,832,864. Also in this case, the problem is the weakening, i.e. strength loss, of denim as a result of sole enzyme treatment employed to impart a stone-washed appearance to the fabric.
• the activity of the cellulase composition depends on the acidity of the application environment. Most generally, the activity is at its highest at slightly acid pHs, even though compositions functioning in a neutral environment may also be employed, and even such cellulase compositions are known as act in alkaline conditions. However, compositions of the latter two types take usually a longer time to act. Buffers known to persons skilled in the art are used to adjust the acidity of cellulase treatment media.
• cellulose-containing textile material refers to textile material composed solely or partly of cellulosic fibres.
• the textile material includes fibre, yarn, woven fabric, knit, or a ready-made garment, in whose manufacture cotton, flax, ramie, jute or man-made cellulosic fibres, for example, viscose, modal, lyocell (e.g. Tencel®) or cupro, have been used as raw material.
• the amount of cellulosic fibre in the textile material has to be at least 30 percent, preferably over 50 percent.
• the present invention is directed to a cellulase composition for treating cellulose- containing textile materials that impart a smooth feel, appearance and softness to the textile.
• the present invention is directed to a cellulase composition for treatment of cellulose-containing textile materials that would not result in significant strength loss of the textile as a result of the treatment.
• the present invention is directed to a cellulase composition for imparting a stone-washed appearance to denims and denim garments without causing significant strength loss to the denims or denim garments.
• the present invention is directed to a treatment medium for cellulose- containing textile materials that may comprise, in addition to the cellulase composition of this invention, for example, surfactants, polymers, buffers, bulk agents, preserv ⁇ atives, stabilisers and/or abrasion agents.
• a treatment medium for cellulose- containing textile materials may comprise, in addition to the cellulase composition of this invention, for example, surfactants, polymers, buffers, bulk agents, preserv ⁇ atives, stabilisers and/or abrasion agents.
• the present invention is additionally directed to a method for treating cellulose- containing textile materials so as to preserve the strength properties, as well as the good appearance and smooth feel of the textile, despite the treatment.
• the described aims of the present invention have been achieved by employing the cellulase composition of this invention in the treatment of cellulose-containing textile materials.
• This cellulase composition is produced using a cellulase solution derived from fungi or bacteria and containing CBH and EG components, the composition of the said solution having been changed so as to contribute to the aims of the invention to be achieved.
• cellulose-containing textile products can be produced that are nice in appearance and feel , show reduced tendency of pilling, and additionally maintain their strength, including also denims and denim garments, to which a stone-washed appearance has been imparted using cellulases.
• Fungi and bacteria can be used as source material for the cellulase composition of the present invention, preferably the cellulase compositions of this invention are derived from fungi, for example from species of Trichoderma, Penicillium, Humicola or Fusarium genera, most preferably Trichoderma reesei is employed as source material.
• the resulting cellulase composition comprising CBH components and various EG components, is not as such applicable to the treatment of cellulose- containing textile materials for producing the desired end result, but, using methods described later in the text, the composition and the ratios between the CBH and EG components are modified so as to obtain a desired composition.
• some other strain than Trichoderma is used as source material, it is possible, in a similar fashion, to modify the ratios of the types of cellulase produced by the employed strain and functionally corresponding to Trichoderma cellulases.
• the cellulase composition for a desired and good end result, it is essential for the cellulase composition not to contain significant amounts or any EGII type endoglucanase of Trichoderma.
• the cellulase composition is derived from Trichoderma, the cellulase composition of the present invention comprises EGI components and possibly small amounts of other EG components, such as EGIII and EGV, and possibly minor quantities of EGII components, and CBHI and perhaps CBHII components.
• the relative ratio of CBHI or EGI to other components can be increased by methods known perse in the art.
• the ratios of the weights of CBH and EG components are not crucial, but it is more important that the amount of EGII type components in the composition is not significant but remains below the normal level, which means that the proportion of EGII should be below 8 weight percent of the total weight of cellulase protein in the cellulase composition. More preferably, the amount of EGII remains below 5 weight percent, and most preferably below 3 weight percent of the cellulase composition, the latter comprising also the alternative of 0 weight percent of EGII.
• the ratio of the weight of CBHI to EG can be greater or equal to 10:1 , but good results have been achieved with the ratio less than 10:1 and even with the ratio 2:1.
• Enzyme activity is one concept applied in determining the properties of enzymes.
• the enzyme activities used in the present patent application are ECU, FPU and MUL, which are defined as follows:
• the endo-1 ,4-beta-glucanase in the sample hydrolyses the hydroxyethylcellulose substrate, and the resulting reducing sugars are assayed spectrophotometrically using a dinitrosalicylic acid reagent (DNS).
• DNS dinitrosalicylic acid reagent
• the cellulase in the sample hydrolyses the filter paper used as a substrate, and the resulting reducing sugars are assayed spectrophotometrically using a DNS reagent.
• the cellobiohydrolase (CBHI) and endoglucanase (EGI) of the sample hydrolyse the 4-methylumbelliferyl-beta-D-lactoside that acts as a substrate, whereby methylumbelliferone is released that can be measured spectrophotometrically.
• the method can be applied in the determination of cellobiohydrolase I (CBHI) activity.
• the method also measures the endoglucanase I (EGI) activity, whose proportion can be determined by inhibiting the activity of cellobiohydrolase using 5 mM of cellobiose.
• One MUL unit is the amount of enzyme activity that in one second under the determination conditions, releases 1 nmol of methylumbelliferone from 4- methylumbelliferyl-beta-D-lactoside (van Tilbeurgh et al., 1988).
• Treatment of cellulose-containing textile materials with the cellulase composition of the present invention imparts a smooth feel, softness and good appearance to the textiles.
• Microscopic pictures of treated cotton-containing fibres additionally show that fibres treated with the composition of this invention have kept their structure better and thus also have remained stronger than fibres that were treated with cellulase compositions comprising considerable amounts of EGII components.
• the treatment of cellulose-containing textile materials with the . cellulase compositions of this invention results in at least 10 percent smaller strength loss and more preferably 20 percent smaller strength loss, than the strength loss measured in textile materials treated with cellulase compositions comprising the original cellulase mixture of Trichoderma. It has also been shown that the treatment of cellulose-containing textile materials with the cellulase compositions of this invention results in at least 15 percent smaller strength loss than the strength loss measured in textile materials treated with cellulase compositions comprising increased level of CBHI component as compared to the original cellulase mixture of Trichoderma.
• Treatment media of cellulose-containing textile materials with compositions disclosed in the present invention may contain, in addition to enzymes, e.g. surfactants, 5 polymers as for example PVA and PVP polymers, buffers as for example citrates, acetates and phosphates for regulating the acidity of the solutions, possibly bulk agents, conventional preserving agents, stabilisers and abrasion agents.
• enzymes e.g. surfactants, 5 polymers as for example PVA and PVP polymers
• buffers as for example citrates, acetates and phosphates for regulating the acidity of the solutions, possibly bulk agents, conventional preserving agents, stabilisers and abrasion agents.
• Suitable cellulase dosages as disclosed in the present invention correspond to the dosages of typical commercial liquid cellulases, as for example Ecostone® L, Ecostone® L 20, Biotouch® L (Primalco Ltd, Biotec, Nurmijarvi, Finland).
• the suitable dosages thus fall within the range of 0.05 to 15 percent, more preferably within 0.5 to 6 percent of the weight of the textile material being treated.
• the suitable enzyme dosages for imparting biofinishing treatment to textile materials depend on the desired result, on the treatment method and the activity of the enzyme product.
• the dosages are about 0.05 to 10 percent, more preferably about 0.5 to 5 percent of the weight of the treated textile material, these dosages corresponding to the dosages of typical commercial liquid cellulases, as for example Biotouch® L, Biotouch® C 601 , Ecostone® L 20 or Ecostone® C 80 (Primalco Ltd, Biotec, Nurmi ⁇ jarvi, Finland).
• the cellulase compositions of this invention can be successfully employed to replace the use of pumice stones.
• the result is a high-quality stone-washed appearance and better strength properties than are achieved in fabrics treated with textile treatment media containing considerable amounts of EGII components.
• the suitable enzyme dosages for imparting a stone-washed appearance to the fabric depend on the desired result, on the treatment method, and on the activity of the enzyme product, and are about 0.05 to 5 percent, more preferably about 0.5 to 2 percent of the weight of the treated fabric, these dosages corresponding to the dosages of typical commercial liquid cellulases, as for example Ecostone® L, Ecostone® L 20, Ecostone® L Plus (Primalco Ltd, Biotec, Nurmijarvi, Finland).
• the pH range for applying the cellulase composition of this invention is dependent on the pH activity profile of the enzyme.
• the pH of the application environment is preferably within the range of 3.5 to 7, more preferably within the range of 4 to 5.5.
• the cellulase composition of the present invention which does not contain considerable amounts of Trichoderma EGII type endoglucanases, can be produced by e.g. fractionation or mutation of the used production strain, or by genetic engineering.
• a cellulase composition free of EGII type endoglucanases or containing a low amount of EGII type endoglucanases can be prepared correspondingly.
• the most convenient way of producing a product with no EGII protein is to delete the gene encoding the EGII protein from the production strain, e.g. from the Trichoderma strain, as described in Example 2a.
• the selected Trichoderma strain or other employed pro ⁇ duction organism can be a wild type strain, or a strain more suitable for use as a production organism developed from the wild type strain by further mutation or genetic engineering.
• suitable strains include, for example, the wild type strain 7. reesei QM6a and the derived mutant strains, e.g. QM9414 and RutC-30, developed for cellulase production, and strains further developed from these, in which e.g. the cellulase and/or hemicellulase level has been further raised, and/or in which the level of produced protease has been lowered.
• VTT-D-79125 and ALK02221 (a mutant strain with a low level of protease production) and their derivatives, as for example the strain overproducing the CBHI enzyme, whose construction is disclosed in Example 2b, are examples of strains that have been further developed.
• the gene encoding the EGII protein (eg/2; Saloheimo et al., 1988; in the publication the gene egl2 is referred to as egl3, which is the original name of the gene) can be deleted from the selected Trichoderma strain by replacing it with a marker gene, or, for example, by a gene encoding EGI (eg/7; Penttila et al., 1986), or by a gene encoding another desired protein in addition to a marker gene.
• the endoglucanase activity of the enzyme mixture produced by the strain is higher than in the enzyme mixture produced by strains from which the egl2 gene is deleted by a marker gene.
• Any marker suitable for Trichoderma can be employed as a marker gene, as for example amdS (e.g. from plasmid p3SR2; Kelly and Hynes, 1985), hygB (e.g. from plasmid pRLM ex 30; Mach et al., 1994) and ble (e.g. from plasmid pAN8-1 , Mattern and Punt, 1988).
• amdS e.g. from plasmid p3SR2; Kelly and Hynes, 1985
• hygB e.g. from plasmid pRLM ex 30; Mach et al., 1994
• ble e.g. from plasmid pAN8-1 , Mattern and Punt, 1988.
• the complementary gene for the auxotrophy concerned can also be employed
• the selected marker gene or the egll gene and a marker gene, are ligated between the 5' and 3' flanking regions of the egr/2 gene forming a targeting plasmid.
• flanking regions homologous recombination can occur and the desired genes can be targeted to replace the egl2 gene.
• the principle of gene replacement is described by Suominen et al. (1993).
• the flank ⁇ ing regions must be long enough, e.g. in Trichoderma at least 1.5 kb in length; examples of flanking regions suitable for replacing cellulase genes have been described by Suominen et al. (1993).
• the necessary flanking regions of the egl2 gene can be isolated from the Trichoderma gene bank, for example by employing an egl2 gene probe that can be synthesised e.g. by the PCR method using the publicised egl2 gene sequence (Saloheimo et al., 1988).
• Strains of EGII-negative phenotype can be selected from among transformants, for example, by analysing the culture media by the Western blot method employing a monoclonal antibody prepared for EGII. Those transformants in which the egl2 gene has been replaced by a marker gene, can be selected also on the basis of their reduced endoglucanase activity as compared to the host strain (ECU activity, Bailey and Nevalainen, 1981). The production level of the CBHI protein can be raised in a selected strain, for example in the Trichoderma reesei strain (examples of applicable 7.
• reesei strains have been given in the foregoing) by mutation or by increasing the copy number of the cbhl gene (Teeri et al., 1983), as is described in Example 2b.
• cbhl promoter also other promoters, suitable for the selected strain, can be used for the expression of the cbhl gene.
• Any marker suitable for Trichoderma may be used as a marker gene in the transformation, as has been described in the foregoing.
• FIG. 1 A microscopic picture of cotton fibres that have not been treated with enzymes.
• Fig. 2 A microscopic picture of cotton fibres treated with the cellulase composition of this invention; EGII has been removed; the CBHI level has been raised as compared to the normal level.
• Fig. 3 A microscopic picture of cotton fibres treated with a cellulase composition comprising CBH components as well as EGI and EGII components; the level of the CBHI component is the same as in the cellulase composition employed in Fig. 2.
• Fig. 4 A microscopic picture of cotton fibres treated with the EGI component.
• Fig. 5 A microscopic picture of cotton fibres treated with the EGII component.
• FIG. 6 A confocal laser scanning microscopic picture of cotton fibres not treated with enzymes.
• Fig. 7 Confocal laser scanning microscopic pictures (A and B) of cotton fibres treated with EGII.
• Fig. 8 eg/2 deletion plasmid pALK175.
• Fig. 9 egl2 targeting plasmid pALK1013 (egll is targeted to replace egl2).
• Fig. 10 Expression plasmid pALK496 for overproduction of CBHI.
• Fig. 12 The effect of a cellulase preparation on the surface cleaning and fuzz removal in woven cotton fabric.
• Top buffer-treated fabric (no enzymes)
• middle the same fabric treated with a preparation containing a normal amount of EGII
• below the same fabric treated by a preparation free of EGII cellulase.
• Enzyme doses 11.2 mg total protein / g fabric, treatment time 1 hour.
• Bleached cotton fibres were cut to a length of 1 mm by scissors.
• the average fibre length was determined by Kajaani FS-200 Fibre analyser.
• the fibres were suspended in a buffer (50 mM Na-acetate buffer, pH 5) and an enzyme was o added on basis of the protein content.
• An enzyme dose of 0.1 to 5.0 mg of protein per 1 gram of fibres was used.
• the incubation temperature was 45°C and time 4 hours.
• the fibres were filtered to a wire in a Buehner funnel.
• the ref ⁇ erence sample was treated in a similar manner without, however, adding any enzyme.
• the fibres were suspended in distilled water of a consistency of 0.2 percent (0.05 g/25 ml). 10 ml of washed glass beads were added to the suspension. The ultrasonic treatment was performed using 50 percent amplitude (Vibra-Cell, Sonics & Materials Inc.), the treatment took 2 minutes. The fibre suspension was kept in ice water during the treatment.
• Samples from the fibre suspension were taken on a glass slide using a pipette.
• the fibres were dyed with Hertzberg colour (SCAN-G 4:90) before microscoping.
• Leica Wild M 10 stereo microscope was used for the microscoping.
• the pictures were taken using a Sony CCD/RGB video camera that was connected to a Mitsubishi CP50E colour printer.
• the fibres were dyed with acridin orange before examination by a confocal laser scanning microscope (Wild Leitz).
• the gene encoding the EGII protein was deleted from 7. reesei ALK02221 and ALKO3760 strains by transforming them by linear fragments isolated from plasmids pALK175 and pALK1013 (Figs 8 and 9).
• ALK02221 is a strain with a low level of protease
• ALKO3760 is a strain overproducing CBHI protein (construction of ALKO3760 is described in Example 2b).
• the fragments contained in the plasmids pALK175 and pALK1013 are described below; the marker gene of the pALK175 fragment replaces the egl2 gene and the marker gene and the eg/7 gene of the pALK1013 fragment replaces the egl2 gene.
• the used DNA methods have been described by Maniatis et al. (1982).
• the gene ble providing fleomycin resistance was used as a marker gene in the plasmids pALK175 and pALK1013.
• the marker gene, and the promoter and terminator used for its expression are derived from plasmid pAN8-1 (about 3.3 kb Bg7ll - Xba ⁇ fragment; Mattern and Punt, 1988).
• the plasmid pALK175 contains a marker gene between the 5' and 3' flanking regions of the 7. reesei egl2 gene.
• the Xho ⁇ - Sac ⁇ fragment of about 1.6 kb (Xho ⁇ is located about 3.8 kb upstream from the egl2 starting codon) was used as the 5' flanking region, and the yAvrll - BamHI fragment of about 1.6 kb (BamYW is located about 1.8 kb downstream from the stop codon of the gene) as the 3' flanking region.
• the employed flanking regions have been isolated from the lambda-clone described by Saloheimo et al. (1988).
• the plasmid pALK1013 contains the 7.
• reesei egll gene (Smal - Seal fragment of about 3.7 kb) for overproduction of EGI.
• the gene is ligated to the SnaBI restriction site of the plasmid pALK175.
• the 7. reesei strains ALK02221 and ALKO3760 were transformed using linear fragments isolated from targeting plasmids, from which fragments the vector sequences had been deleted (pALK175: EcoRI + Ba HI, about 6.5 kb; pALK1013: EcoRI + BamHI, about 10.2 kb).
• EGII-negative strains were selected by analysing the culture media by the Western blot method using the monoclonal antibody prepared for EGII.
• the ECU and FPU activities, and, using the ELISA method, the amounts of CBHI, CBHII and EGI proteins were analysed in the culture media of the selected transformants.
• the replacement of the egl2 gene in the genomes of the transformants was confirmed by the Southern blot analysis.
• the ECU activity of the enzymes produced by the strains was about 50 to 60 percent of the ECU activity of the enzymes produced by the host strain.
• the FPU activities of the EGII deletion strains showed a decline of about 20 to 30 percent as compared to the FPU activity of the host strain. 2b. Construction of a strain overproducing the CBHI protein
• the expression plasmid pALK496 constructed for overproduction of CBHI is shown in Fig. 10.
• the promoter cbhl, the gene cbhl (Teeri et al., 1983) and the cbhl terminator used to construct the plasmid are derived from the 7 reesei strain QM6a.
• the cbhl fragment (Stu ⁇ - Nru ⁇ ) of 4.95 kb contains a promoter region of approximately 2.2 kb, a cbhl gene (about 1.6 kb), and a terminator region of approximately 0.7 kb.
• An acetamide gene (amdS) was used as a marker gene in the plasmid.
• amdS encodes acetamidase whereby transformants possessing this gene are capable of growing on acetamide as a sole nitrogen source. This property was employed in the selection of the transformants.
• An acetamide fragment (Spe ⁇ - Xba ⁇ ) of 3.1 kb derived from plasmid p3SR2 (Kelly and Hynes, 1982) was used to construct the pALK496 plasmid. The said fragment contains an acetamide gene, a promoter and a terminator.
• the amdS and cbhl fragments were ligated between the 5' and 3' flanking regions of the gene eg/7.
• flanking regions Using the flanking regions, the desired genes can be targeted to replace the eg/7 gene, when necessary.
• the flanking regions used in the plasmid i.e. 1.8 kb 5' (Seal - Stu ⁇ ) and 1.6 kb 3' (Ba HI - Xho ⁇ ), are derived from the 7. reesei QM6a strain.
• the expression plasmid was constructed employing standard DNA methods (Maniatis et al., 1982).
• the 7. reesei strain ALK02221 (possessing a low level of protease activity) was transformed by linear fragments isolated from an expression plasmid, from which fragments vector sequences had been removed.
• the employed transformation method and selection medium have been described by Penttila et al. (1987) and Karhunen et al. (1993).
• the transformants were treated as described in the said references and grown on a cellulase-inducing lactose-based medium as described in Example 2a.
• the activity in the culture medium of the transformants hydrolysing 4- methylumbelliferyl-beta-D-lactoside (MUL) van Tilbeurgh et al., 1988 was measured.
• the increased activity indicated an increase in the CBHI production level. Also the FPU activity (IUPAC, 1984), the amount of total protein (Lowry et al., 1951), and, by the ELISA method (Buehler, 1991), the amounts of CBHI, CBHII and EGI proteins were analysed in the culture media of the selected transformants. The structures of the genomes of the selected transformants were confirmed by the Southern blot analysis.
• the production of CBHII and EGI proteins of the strain was about 25 percent lower than that of the host strain.
• the preparation was made mixing the following components:
• Component 1 The cellulase preparation of the present invention containing no EGII type endoglucanase.
• Component 2 A cellulase preparation containing more EGI type endoglucanase than normal, but no CBHI type cellulase (Karhunen T., et al., 1993). A product containing below 3 percent EGII protein of the total cellulase protein amount (cf. Test 4 in Example 4) was obtained mixing the above components in athe following ratio:
• the effect of the cellulase composition of the present invention on the properties of cellulose-containing textile materials was investigated by treating fabric samples with methods corresponding to normal cellulase treatment, and by measuring strength values of the fabrics after treatment and by estimating the washing results.
• An enzyme preparation was added which contains all CBH and EG enzyme components (normal amount of EGII) resulting in the total protein content of the treatment medium being about 0.05 mg/ml (reference),
• a cellulase preparation in accordance with the invention was added which does not contain any EGII type cellulase. Measured as FPU activity, the enzyme doses were similar to those in Test 2.
• a cellulase preparation in accordance with the invention and prepared as in Example 3 was added, the preparation containing below 3 percent of EGII type cellulase.
• the enzyme activities measured as ECU and FPU activities were on the same level as when using the doses described in Test 2.
• Textile material (100% cotton fabric and 100% cotton denim) was treated according to the textile washing process as described in Example 4 using cellulase preparations in accordance with Tests 5 and 6.
• a cellulase preparation was added which contained all CBH and EG enzyme components (the amount of EGII was normal) and in which the amount of CBHI was raised over the normal level by genetic engineering.
• the total protein content of the treatment medium was about 0.03 mg/ml.
• the enzyme preparation of the present invention was added that was free of EGII type cellulase.
• the dosing was adjusted so as to settle the ECU and FPU activities of the treatment medium on the same level as in Test 5.
• the bursting strength of the cotton fabrics was measured as described in Example 4. The results are given in Table 2.
• Test 6 in which the enzyme composition of the present invention was used, the strength loss was smaller than in Test 5, in which the employed enzyme composition contained a raised level of the CBHI component and the proportion of the EGII component was normal. Both tests imparted a high-quality stone-washed appearance to denims.
• denim jeans The treatment of denim jeans was performed using a washing machine with a capacity of approximately 410 I (900 lbs). In each test, approximately 145 kg (320 lbs) of jeans were washed.
• washing process 1. Prewash to remove starch, alpha-amylase preparation temperature 70°C, washing time 7 minutes liquid ratio 8:1 amount of surfactant 0.45 kg (1 lb)
• An enzyme preparation which contains all CBH and EG cellulase , components and in which the amount of CBHI is raised by genetic engineering and the amount of EGII is normal, i.e. 1.5 percent of the weight of the fabric.
• An enzyme preparation which contains all CBH and EG cellulase components (the amount of EGII was normal), 0.75 percent of the weight of the fabric + 100 percent of the weight of the fabric was pumice stone (an ordinary industrial process).
• the enzyme preparation according to this invention and free of EGII type cellulase 5 was used.
• the dosage was adjusted so as to obtain an ECU activity level in the treatment medium similar to that in Test 1.
• the strength loss was the smallest, smaller than in cases where the jeans were treated with enzymes and pumice stones, or with an enzyme preparation with a raised level of CBHI.
• the treatment conditions were as follows:
• An enzyme preparation which contains all CBH and EG cellulase components, and in which the amount of CBHI is raised by genetic engineering, and the amount of EGII is normal. 2.
• the enzyme preparation disclosed above but free of EGII type cellulase was used. The preparations were dosed so that equal amounts of protein were used.
• the surface cleaning effect of the enzyme preparations was estimated by measuring the weight loss of the fabric, by analysing the visual appearance, and by the Martindale Rubbing Method (SFS 4328). Weight loss of the cotton swatches was determined as follows: The swatches were preconditioned before and after the cellulase treatment in an atmosphere of 22°C and 50% RH for 12 hours and weighed. The percentage of the weight loss was calculated:
• Percentage of the weight loss (weight of the swatch before treatment - weight of the swatch after treatment) x 100/weight of swatch before treatment
• Figure 11 shows how the weight loss of the fabric increases as a function of the enzyme dose at different treatment times.
• the weight loss profile is similar for the two cellulase preparations. In commercial processes weight loss of 3-5% usually gives a proper wearing effect.
• Figures 12 shows photographs of the surface of the fabrics before and after enzyme treatment. Treatment of the fabric with either of the studied cellulase preparations results in good surface cleaning effect and fuzz removal. Due to fuzz removal, also the texture of the fabric becomes more visible.
• the tearing strenght of the swatches was determined by the Elmendorf-method (SFS 3982).
• the strength loss of the examined fabrics after biofinishing with the two cellulase mixtures in Launder-Ometer is shown in figure 14.
• the cellulase preparation free of EGII gives the lower strength loss as a function of enzyme dosage and weight loss at both of the treatment times.

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