WO1996034945A1

WO1996034945A1 - Cellulase composition for treatment of cellulose-containing textile materials

Info

Publication number: WO1996034945A1
Application number: PCT/FI1996/000248
Authority: WO
Inventors: Arja Sisko Kaarina Miettinen-Oinonen; Minna Johanna Elovainio; Pentti Juhani Ojapalo; Marja Tuulikki Paloheimo; Pirkko Liisa Suominen; Jaakko Juhani Pere; Anne Margit ÖSTMAN
Original assignee: Röhm Enzyme Finland OY
Priority date: 1995-05-05
Filing date: 1996-05-06
Publication date: 1996-11-07
Also published as: FI952165A0; EP0824584A1; AU5649596A

Abstract

The present invention is directed to a new cellulase composition for treatment and finishing of cellulose-containing textile materials. The strength properties of cellulose-containing textile materials have remained better in fabrics treated with the compositions of the present invention than the corresponding properties in cellulose-containing textile materials treated with earlier cellulase compositions. The main feature of the new composition is that it does not contain significant amounts of EGII-type endoglucanases.

Description

CELLULASE COMPOSITION FOR TREATMENT OF CELLULOSE-CONTAINING TEXTILE MATERIALS

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.

Cellulase treatment of cellulose-containing textile materials during their manufacture or finishing is known per se in the art. The enzymatic treatment for finishing of cellulose-containing textile materials is called biofinishing. 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.

Additionally, 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.

Significant strength loss of the fabric as a result of cellulase treatment is a problem associated with cellulase treatment. The strength loss is caused by cellulase-induced hydrolysis of beta-1 ,4-glucosidic bonds of cellulose, which in turn results in partial degradation of cellulosic polymer, and further, can result in strength loss of the fabric. A method for treating cotton fabrics, prior to dyeing and finishing, with cellulase solution in order to remove lint and loose surface fibres to impart a better appearance to the fabric is disclosed in the US Patent No. 5,232,851. The employed cellulase can be produced, for example, by Trichoderma reesei, T. koningii, Penicillium sp or Humicola insolens species. In the given examples, CYTOLASE 123 -cellulase (Genencor Int.) was used with no detailed composition given in the said publication. In addition to cellulase, 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.

In the treatment of fabrics, cellulase derived from fungi, for example from Trichoderma reesei, is generally employed, such cellulase being composed of cellobiohydrolase (CBH), endoglucanase (EG) and beta-glucosidase (BG) type components. The 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 isolation of cellulase components from fungi is known in the art. See, for example, Bhikhabhai, R. et al., (1984), Saloheimo, M., et al., (1988), Wood et al., (1988), Bhat, K.M. et al., (1989) and Schulein (1988).

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. The US Patent No. 5,120,463 discloses 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 (FI-945122) 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. The application mentions that the tensile strength of treated fabric is at least about 50 percent of the tensile strength of untreated fabric. No test results, however, are given.

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.

The stone-wash was traditionally performed using so-called pumice stones. However, the use of 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.

It is known in the art that 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.

In view of the above, the primary problem of using cellulase compositions in the treatment and finishing of cellulose-containing textile materials has thus been the strength loss of the fabric as a result of the cellulase treatment.

The term "cellulose-containing textile material " as used in the present invention 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. When using synthetic man-made fibres, 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.

In particular, 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.

Additionally, 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.

Further, 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.

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.

It has unexpectedly been found out that by employing, in the treatment or finishing of cellulose-containing textile materials, a cellulase composition which is characterised by claim 1 of the present invention, 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. When 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.

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. If 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. In the cellulase composition of the present invention, however, 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. In the cellulase composition of the present invention 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:

ECU

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). One unit of endo-1 ,4-beta-glucanase is defined as the amount of enzyme producing one nmole of reducing sugars as glucose in one second (1 ECU = 1 nkat), (Bailey, M. and Nevalainen, H., 1981).

FPU

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 filter paper degrading activity is described as FPU units. The calculation is based on the definition of the International Unit (IU). 1 IU = 1 micromol min.^"1 of product formed (reducing sugars as glucose) (IUPAC, 1984).

MUL

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.

In regard to the above, it has been possible to show that the strength properties of cellulose-containing textile materials, i.e. bursting strength, breaking strength and tearing strength have remained better in textile materials treated with the compositions of this invention than the corresponding properties in cellulose- containing textile materials treated with earlier cellulase compositions. It has additionally been shown that cellulose-containing textile materials treated with the cellulase composition of this invention have lost less than 30 percent of their strength properties as compared to the cellulose-containing textile materials treated in the same conditions without cellulases. More preferably textile materials treated with the cellulase composition of this invention have lost less than 20 percent of their strength properties, and most preferably 10 percent as compared to the cellulose-containing textile materials treated in the same conditions without cellulases.

It has been shown that 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.

The dosage of cellulase products in a solution depends on the desired result, o application, the activity of the cellulase product etc. 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).

For imparting a stone-washed appearance to denims, 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. When enzymes functioning at acid pHs are being employed, 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. As for Trichoderma reesei, 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.

A general description of the methods for deleting the gene encoding the EGII protein, and of the method for increasing the production level of the CBHI protein in Trichoderma reesei is described in the following. Applying the same method, it is possible to change the ratios of cellulases also in other species and, for example, to delete genes encoding Trichoderma EGII type cellulase(s).

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. When the egl2 gene is replaced by the egll gene and 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_ex30; Mach et al., 1994) and ble (e.g. from plasmid pAN8-1 , Mattern and Punt, 1988). For auxotrophic strains, the complementary gene for the auxotrophy concerned can also be employed as a marker.

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. Using the 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). For the targeting frequency to be satisfactorily high, 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. In addition to the 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.

The sources referred to in the above description are given in the reference list.

The following examples and figures provide further details of the preparation of the cellulase composition of the present invention, and the effect of the cellulase composition on the properties of cellulose-containing textile materials.

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. 11. Weight loss of fabrics treated with enzymes.

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, and 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.

Fig. 13. Pilling tendencies in woven cotton fabric after cellulase treatment in Launder Ometer for 1 hour and 2 hours.

Dosage:

1. treated with buffer, no cellulases added

2. a normal amount of EGII, 2.8 mg total protein / g fabric

3. a normal amount of EGII, 11.2 mg total protein / g fabric 4. free of EGII, 2.8 mg total protein / g fabric

5. free of EGII, 11.2 mg total protein / g fabric Fig. 14. Tearing strength in relation to the weight loss of the cellulase treated fabrics.

Example 1

The cotton fibres shown in Figures 1 to 7 were treated and microscoped as described below:

Enzyme treatment

Bleached cotton fibres (Kolmiset Oy) 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. After the treatment, 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.

Ultrasonic treatment

After the enzyme treatment, 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.

Microscoping

Samples from the fibre suspension were taken on a glass slide using a pipette. For a better contrast, 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.

For a more detailed microscopic analysis, the fibres were dyed with acridin orange before examination by a confocal laser scanning microscope (Wild Leitz).

The detrimental effect to fibre structures of the EGII enzyme can clearly be seen in the pictures. As a result of the hydrolytic activity of the EGII cellulase and a light mechanic treatment, the wall structure of the cotton fibres is locally fibrillating and, in a worst case, breaking. Due to damaged wall structures, the fibres suffer strength losses, which is further reflected as lowered strength properties of woven fabrics. On the other hand, fibres or fabrics not treated with preparations containing EGII cellulase kept their structures better and thus were also stronger. Example 2

Publications referred to in the examples are listed in the attached list of references.

2a. Deletion of the αene encoding the EGII protein

For the preparation of the cellulase composition of the present invention, 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). In addition to the flanking regions of egl2 and the marker gene (as in pALK175), 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).

The employed transformation method has been described by Penttila et al. (1987) and Karhunen et al. (1993). An MnR medium was used as a selection medium (Durand et al., 1988). From selection plates containing fleomycin, transformants were picked to selection slants and purified on selection plates through spores, as has been described by Penttila et al. (1987). The transformants were stabilised by growing them on fleomycin slants for three cycles before moving them to PD slants (Potato Dextrose agar, Difco). From the PD slants the transformants were inoculated to a rich lactose-based medium inducing cellulases (Suominen et al., 1993), where they were grown at 30°C for 7 days (250 rpm).

From among the transformants, 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.

After the deletion of the egl2 gene by the marker gene, 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 ECU activity of the strains from which the egl2 gene was deleted and replaced by the eg/7 gene and a marker gene, was on the same level or higher than in 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. Wild type strains are incapable of growing with acetamide as their sole source of nitrogen. 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. In the expression plasmid, the amdS and cbhl fragments were ligated between the 5' and 3' flanking regions of the gene eg/7. 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 MUL activity of the Trichoderma transformant, constructed as described in this Example and overproducing CBHI and containing all other cellulases, had increased 1.4-fold as compared to the activity of the host strain. The production of proteins of the strain was about 10 percent higher than that of the host strain, the FPU activity was similar in both strains. The level of produced CBHI protein rose as compared to the host strain. The production of CBHII and EGI proteins of the strain was about 25 percent lower than that of the host strain.

Example 3

Preparation of an enzyme product that contains more than 0 percent, but not a significant amount (i.e. below 8 percent of the total amount of cellulase protein) of EGII type endoglucanase.

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:

88 volume percent of a preparation in accordance with component 1 showing enzyme activities of 7000 ECU/ml and 60 FPU/ml.

12 volume percent of a preparation in accordance with component 2 showing enzyme activities of 54000 ECU/ml and 17 FPU/ml.

Example 4

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.

The reference results were obtained treating the fabrics in a similar fashion but without enzymes and using commercial enzyme products.

100 percent cotton fabric and 100 percent cotton denim were used in the tests. A semi-industrial drum washer (Esteri 20 HS-P) was used with an intake of water of about 100 I.

The fabrics were washed and treated as follows:

1. Prewash to remove starch, temperature 60°C, washing time 10 minutes. 1 ml of an alpha-amylase preparation

(Ecostone® A, Primalco Ltd, Biotec, Nurmijarvi) per 1 I of water was used in the prewash. 2. Cellulase treatment, temperature 50°C, treatment time 45 minutes.

Various cellulase preparations with described doses (Tests 1 to 4) were used, pH 5 adjusted by 80 percent acetic acid, liquid ratio of about 20:1.

3. Wash with a detergent, temperature 40°C, washing time 10 minutes.

4. Drying

TESTS

Test 1. Treatment without added enzyme (reference),

Test 2.

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),

Test 3.

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.

Test 4.

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.

Strength measurements

After treatment, the following strength values were measured for cotton fabrics: 5 Breaking load and elongation (according to the SFS Standard 2983)

Tearing strength (according to the SFS Standard 3981)

Bursting strength (no standard, measured by the Adamel-

Lhomargy Burst Tester EC 07 following the l o instructions of the manufacturer)

The results are given in Table 1. The breaking load and elongation as well as the bursting strength are measured for the warp and weft.

15 The results show that when the cellulase compositions of the present invention (Tests 3 and 4) are used in the treatment of cellulose-containing textile materials, the strength loss is smaller than in cases where the textiles are treated with a cellulase composition containing a normal amount of the EGII type component produced by Trichoderma (Test 2) as compared to textiles not treated with cellulase (Test 1).

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Table 1. Strength properties of treated cellulose-containing textile materials (Tests 1 to 4).

TEST 1 TEST 2 TEST 3 TEST 4

Breaking load, warp (N) 481 387 410 410

Elongation, warp (%) 16.7 14.2 14.9 14.8

Breaking load, weft (N) 458 356 375 353

Elongation, weft (%) 28.1 26.2 26.3 26.9

Tearing strength, warp (N) 11.1 7.0 8.1 7.2

Tearing strength, weft (N) 10.8 7.2 8.5 7.7

Bursting strength (kP) 702 555 573 562

Employing any of the cellulase treatments (Tests 2 to 4), a high-quality stone-washed appearance was imparted to denims.

Example 5

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.

Test 5

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.

Test 6

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. After treatment, the bursting strength of the cotton fabrics was measured as described in Example 4. The results are given in Table 2.

Table 2. Strength measurements of Tests 5 and 6.

Test 5 Test 6

Bursting strength (kPa) 795 851

In 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.

Example 6

A test of denims on an industrial scale to impart a stone-washed appearance to jeans (denim) employing three different cellulase compositions (Tests 7 to 9).

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)

2. Rinsing 2 times 3. Stone-wash a cellulase preparation with or without stones temperature 60°C, washing time 45 to 70 minutes pH 4.6 (acetate buffer) liquid ratio 8:1 amount of surfactant 0.45 kg (1 lb)

4. Rinsing and bleaching

TESTS

Test 7

An enzyme preparation was used 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.

Test 8

An enzyme preparation was used 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).

Test 9

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 results showed that the cellulase composition of the present invention (Test 9) created a stone-washed appearance which was of as high a standard as the effect o achieved by the present method (Test 8). The tearing strengths of the treated jeans are shown in Table 3.

Table 3. Tearing strengths of jeans.

TEST 7 TEST 8 TEST 9

Tearing strength, warp (N) 27.5 25.3 30.9

Tearing strength, weft (N) 21.7 24.3 25.9

Employing the composition of the present invention (Test 9), 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.

Example 7

The biofinishing effects of the cellulase composition of the present invention on cotton fabric were studied on a laboratory scale.

100 percent cotton woven fabric was subjected to treatment with two cellulase preparations in laboratory-scale washings in Launder-Ometer (Atlas LP2).

The treatment conditions were as follows:

1. Prewash to remove starch in Cylinda 14003 washing machine

10 ml of Ecostone A200 α-amylase preparation, program 2, 60°C

2. Cellulase treatment

Two swatches of 28x25 cm / 1.2 I container 50°C, washing time 1 and 2 hours, liquid ratio 20:1 0.05 M Na-citrate buffer pH 5.2

3. Washing of the swatches with an alkaline detergent and rinsing with water, drying. Cellulase preparations:

1. An enzyme preparation was used 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.

In order to evaluate the efficiency of the two cellulase preparations to reduce pill formation, the fabrics treated with enzymes were subjected to the Martindale Rubbing Method (200 cycle abrasion). The test results were evaluated by a panel on a scale of 1 to 5. The cellulose-containing textile material treated in the same conditions without cellulases was given the value 1. Figure 13 illustrates the reduction of pill formation in cotton fabric treated with the two tested cellulase preparations. The cellulase preparation free of EGII is as efficient in pilling removal as the preparation with normal as amount of EGII.

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.

References referred to in the general description and the examples are listed in the following:

Bailey, M.J. and Nevalainen, K.M.H. 1981. Induction, isolation and testing stable Trichoderma reesei mutants with improved production of solubilizing cellulase. Enzyme Microb. Technol. 3:153-157.

Bhat, K.M., McCrae, S.I. and Wood, T.M. 1989. The endo-1-4-beta-d-glucanase system of penicillium-pinophilum cellulase isolation purification and characterization of five major endoglucanase components. Carbohydrate Research 190:279-297.

Bhikhabhai, R., Johansson, G. and Pettersson, G. 1984. Isolation of Cellulolytic Enzymes from Trichoderma reesei QM9414. Journal of Applied Biochemistry 6:335- 345.

Bϋhler, R. 1991. Double-antibody sandwich enzyme-linked immunosorbent assay for quantitation of endoglucanase I of Trichoderma reesei. Appl. Environ. Microbiol. 57:3317-3321. Durand, H., Baron, M,, Calmels, T. and Tiraby, G. 1988. Classical and molecular genetics applied to Trichoderma reesei for the selection of improved cellulolytic industrial strains. In: Biochemistry and Genetics of Cellulose Degradation, Academic Press, New York, pp. 135-151.

IUPAC Commission on Biotechnology. 1984. Measurement of Cellulase Activities, Ghose, T.K.

Karhunen, T., Mantyla, A., Nevalainen, K.M.H. and Suominen, P.L. 1993. High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction. Mol. Gen. Genet. 241 :515-522.

Kelly, J.M. and Hynes, M.J. 1985. Transformation of Aspergillus niger by the amdS gene of Aspergillus nidulans. EMBO J. 4:475-479.

Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

Mach, R.L., Schindier, M. and Kubicek, C.P. 1994. Transformation of Trichoderma reesei based on hygromycin B resistance using homologous expression signals. Curr. Genet. 25:567-570.

Maniatis, T., Fritsch, E.F. and Sambrook, J. 1982. Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory.

Mattern, J.E. and Punt, P.J. 1988. A vector of Aspergillus transformation conferring fleomycin resistance. Fungal Genet. Newslett. 35:25.

Penttila, M., Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and Knowles, J. 1986. Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene. Gene 45:253-263.

Penttila, M., Nevalainen, H., Rattό, M., Salminen, E. and Knowles, J. 1987. A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61 :155-164.

Saloheimo, M., Lehtovaara, P., Penttila, M., Teeri, T.T., Stalberg, Johansson, G., Pettersson, G., Clayessens, N., Tomme, P. and Knowles, J.K.C. 1988. EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63:11-21.

Schulein, M. 1988. Cellulases of Trichoderma reesei. Methods in Enzymology 160:234-242.

Suominen, P.L., Mantyla, A., Karhunen, T., Hakola, S. and Nevalainen, H. 1993. High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes. Mol. Gen. Genet. 241 :523-530.

Teeri, T., Salovuori, I. and Knowles, J. 1983. The molecular cloning of the major cellulase gene from Trichoderma reesei. Bio/technology 1 :691-699.

van Tilbeurgh, H., Loontiens, F.G., De Bruyne, C.K. and Clayessens, M. 1988. Fiuorogenic and chromogenic glycosides as substrates and ligands of carbohydrases. Methods in Enzymology 160:45-59.

Wood, T.M., McCrae, S.I., Wilson, C.A., Bhat, K.M. and Gow, L.A. 1988. Aerobic and anaerobic fungal cellulases, with special reference to their mode of attack on crystalline cellulose. FEMS Symposium No. 43, Biochemistry and Genetics of Cellulose Degradation, pp. 31-52.

Claims

1. A cellulase composition for treatment of cellulose-containing textile materials during their manufacture or finishing comprising CBH type (cellobiohydrolase type) and EG type (endoglucanase type) components and resulting in good biofinishing effects and/or in a good stone-washed appearance , characterised in that said cellulase composition does not contain significant amounts of EGII type components and that said treatment with said cellulase composition does not cause significant strength loss to the textile materials.

2. The cellulase composition according to claim 1 characterised in that said composition comprises one or more CBH type and EG type components with the proportion of EGII type components being below 8 weight percent, preferably 5 weight percent and most preferably below 3 weight percent of the total cellulase protein content of the composition.

3. The cellulase composition according to claims 1 or 2 characterised in that said composition contains no EGII type components.

4. The cellulase composition according to claims 1 to 3 characterised in that said cellulase composition is derived from fungi or bacteria, for example from fungi of Trichoderma, Penicillium, Humicola or Fusarium genera, preferably from Trichoderma reesei species.

5. The cellulase composition according to claims 1 to 4 characterised in that the amount of CBHI and/or EGI type components in said composition is increased compared to its/their amount in the original cellulase mixture of Trichoderma.

6. The cellulase composition according to claim 5 characterised in that said cellulase composition has been obtained using a CBHI and/or EGI overproducing Trichoderma reesei strain.

7. A treatment medium for cellulose-containing textile materials characterised in that said treatment medium contains a cellulase composition according to any one of claims 1 to 6.

8. The treatment medium according to claim 7, characterised in that the treatment of cellulose-containing textile materials with the said medium results in a strength loss smaller than 30 percent, more preferably a strength loss smaller than 20 percent, most preferably smaller than 10 percent as compared to the cellulose- containing textile materials treated in the same conditions without cellulases.

9. The treatment medium according to claim 7, characterised in that the treatment of cellulose-containing textile materials with the said medium 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 compositions comprising the original cellulase mixture of Trichoderma.

10. The treatment medium according to claim 7, characterised in that the treatment of cellulose-containing textile materials with the said medium results in at least 15 percent smaller strength loss than the strength loss measured in textile materials treated with compositions comprising increased level of CBHI component as compared to the original cellulase mixture of Trichoderma.

11. A treatment medium for the biofinishing of cellulose-containing textile materials, characterised in that said treatment medium contains a cellulase composition according to any one of claims 1 to 6.

12. A treatment medium for imparting a stone-washed appearance to denim fabrics or garments characterised in that said treatment medium contains a cellulase composition according to any one of claims 1 to 6.

13. A treatment medium according to claim 11 or 12, characterised in that 5 said treatment medium contains surfactants, polymers, buffers, bulking agents, preservatives, stabilisers and/or abrasion agents.

14. A method for treatment of cellulose-containing textile materials characterised in that the treatment medium according to claims 7 to 13 is used in the l o treatment or finishing of cellulose-containing textile materials.

15. The method for treatment of cellulose-containing textile materials according to claim 14, characterised in that the proportion of cellulosic fibre in the textile material is at least 30 percent, more preferably 50 percent.

15

16. The method for treatment of cellulose-containing textile materials according to claims 14 and 15, characterised in that the cellulosic fibre in the cellulose-containing textile material is cotton, flax, ramie, jute, viscose, modal, lyocell, or cupro.

20

17. The method for treatment of cellulose-containing textile materials according to claims 14 to 16, characterised in that said treatment with said medium results in a strength loss smaller than 30 percent, more preferably a strength loss smaller than 20 percent, most preferably a strenght loss smaller than 10 percent as

25 compared to the cellulose-containing textile materials treated in the same conditions without cellulases.

18. The method for treatment of cellulose-containing textile materials according to claims 14 to 16, characterised in that said treatment with said medium

30 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 compositions comprising the original cellulase mixture of Trichoderma.

19. The method for treatment of cellulose-containing textile materials according to claims 14 to 16, characterised in that said treatment with said medium results in at least 15 percent smaller strength loss than the strength loss measured in textile materials treated with compositions comprising increased level of CBHI component as compared to the original cellulase mixture of Trichoderma.