JP2654562B2 - Method for treating cotton-containing fabrics with cellulase - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to improved methods of treating cotton-containing fabrics with cellulase, and to fabrics produced by these methods. In particular, the improved method of the present invention relates to contacting a cotton-containing fabric with an aqueous solution containing a fungal cellulase composition that is substantially free of all CBH type I cellulase components. When the cotton-containing fabric is treated with such a solution, the resulting fabric is of high quality compared to the untreated fabric, for example, in terms of feel, appearance, and / or softness, and the like. The fabric also
The loss of strength is reduced as compared to a fabric treated with a cellulase composition containing a higher concentration of the CBH type I component.

2. Prior Art During or shortly after the production of the fabric, the cotton-containing fabric can be treated with cellulase to give the desired properties to the fabric. For example, in the textile industry, cellulases are used to improve the feel and / or appearance of cotton-containing fabrics, to remove surface fibers from cotton-containing knits, and to provide the appearance of stone wash to cotton-containing denims and the like. ing.

In particular, Japanese Patent Application Nos. 58-36217 and 58-54032,
And Oishi et al., "Reforming Cotton Fabrics with Cellulase" and JTN December 1988, "New-Weight Loss Treatment to Soften the Feel of Cotton Fabrics", respectively. It discloses providing an improved feel. Generally, this cellulase treatment removes fluff and / or surface fibers, which is believed to reduce the weight of the fabric. The combination of these effects gives the fabric an improved feel, ie a silky fabric feel.

In addition, it is conventionally conventional to treat cotton-containing knits with a cellulase solution under stirring and cascade conditions, for example by using a jet, for the purpose of removing broken fibers and yarns which are usual in these fabrics. Known to the trader.
In such treatments, buffers are not usually used, as the buffers are believed to adversely affect the dye shade with the selected dye.

It is even further known in the prior art to treat cotton-containing fabrics with cellulase solutions under stirring and cascade conditions. When so treated, the cotton-containing fabric exhibits improved feel and appearance as compared to the untreated fabric.

Finally, it is hitherto known that the treatment of cotton-containing dyed denim with a cellulase solution under stirring and cascade conditions, ie in a rotating drum washing machine, gives the denim a "stone wash" appearance.

A common problem associated with treating such cotton-containing fabrics with a cellulase solution is that the treated fabrics exhibit a significant loss of strength as compared to untreated fabrics. Cellulase hydrolyzes cellulose (β-1,4-glucan linkages), which in turn can result in fragmentation of the cotton macromolecule, resulting in a loss of strength. As the cotton polymer gradually divides (divides), the tensile strength of the fabric decreases.

Since methods involving stirring and cascading of the cellulase solution over the cotton fabric require shorter reaction times,
These methods are believed to provide cotton-containing fabrics with reduced strength loss compared to cellulase treatment methods that do not include agitation and cascade. In any case, such a method still results in significant strength loss.

Therefore, such a cellulase treatment method is improved to achieve the desired enhanced performance in the treated cotton-containing fabric produced by treatment with cellulase compared to the untreated fabric, but to provide reduced strength loss. It is particularly desirable.

In addition, fungal sources of cellulases are known to secrete very large amounts of cellulases, and furthermore, fermentation methods for such fungal sources and isolation and purification methods for isolating cellulases are known in the art. As is known, it is particularly advantageous to use such fungal cellulases in methods for improving the feel and / or appearance.

SUMMARY OF THE INVENTION The present invention relates to a method for treating cotton-containing fabrics with fungal cellulases, the method comprising the steps of: providing a fungal cellulase comprising one or more EG-type components and containing a sufficiently low concentration of CBH type I components; It is based on the finding that it can be improved by using the composition. Surprisingly, the EG-type component provides enhanced texture to the treated fabric in terms of feel, appearance, softness, color, and / or stone wash appearance as compared to the fabric prior to treatment with the cellulase composition. Discovered that In addition, it has been found that a significant portion of the strength loss in the treated fabric is due to the CBH type I component combined with the EG type component. Thus, in the present invention, the cellulase composition used to treat the cotton-containing fabric is made to contain a sufficiently low concentration of the CBH type I component to be strength loss resistant.

In one aspect of the method in view of the above, the present invention is based on an improved method of treating a cotton-containing fabric with a fungal cellulase composition, wherein the improvement comprises one or more EG-type components. And using a fungal cellulase composition comprising one or more CBH type I components, wherein the cellulase composition has a protein weight ratio of all EG type components to all CBH type I components of greater than 5: 1. Having.
Here, “greater than 5: 1” refers to those excluding the case where CBH type I is not included. That is, the CBH type I component is necessarily present in the cellulase composition of the present invention. In a preferred embodiment, the fungal cellulase composition used herein consists of one or more EG-type components and one or more CBH-type components, wherein the cellulase composition comprises all CBH-type components greater than 5: 1. Has the protein weight ratio of all EG-type components to In yet another preferred embodiment, the fungal cellulase composition comprises at least about 10 weight percent, preferably at least about 20 weight percent, of the EG-type component, based on the total weight of the protein in the cellulase composition.

In another aspect of the method, the present invention is based on an improved method of treating a cotton-containing fabric with an aqueous fungal cellulase solution, wherein the method comprises conditions that produce a cascading effect of the cellulase solution across the fabric. Agitating the cellulase solution below, wherein the improvement comprises using a fungal cellulase composition consisting of one or more EG-type components and one or more CBH I-type components, wherein the cellulase composition comprises: It has a protein weight ratio of all EG type components to all CBH type I components of greater than 5: 1. In a preferred embodiment, the fungal cellulase composition used herein consists of one or more EG-type components and one or more CBH-type components, wherein the cellulase composition is greater than 5: 1.
It has the protein weight ratio of all EG type components to all CBH type components. In yet another preferred embodiment, the fungal cellulase composition comprises at least about 10 weight percent, preferably at least about 20 weight percent, of the EG-type component, based on the total weight of the protein in the cellulase composition.

The cotton-containing fabric treated by the method of the present invention shows reduced strength loss as compared to the fabric treated with the fungal cellulase composition containing a higher amount of CBH type I component, but as compared to the fabric before treatment. With enhanced quality as expected.
The reduced strength loss justifies that the method of the invention is strength loss resistant.

In terms of its composition, the invention is based on the cotton-containing fabric treated in the method of the invention described above.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of the structure of pΔCBHIpyr4.

FIG. 2 shows pΔCB at the cbh1 locus on one T.reesei chromosome.
T.reesei by integration of a large EcoR I fragment from HIpyr4
The figure explaining deletion of a gene.

FIG. 3 shows T. transformed with EcoRI cut pΔCHBIpyr4 after Southern analysis using ³² P-labeled pΔCBHIpyr4 as a probe.
Figure 9 is an autoradiograph of DNA from reesei strain GC69. The size of the molecular weight markers is shown in kilobase pairs on the left side of the figure.

FIG. 4 shows T.rees transformed with EcoRI cut pΔCBHIpyr4 after Southern analysis using ³² P-labeled pIntCBHI as probe.
Figure 9 is an autoradiograph of DNA from ei strain GC69. The size of the molecular weight markers is shown in kilobase pairs on the left side of the figure.

FIG. 5 is an isoelectric focusing gel showing proteins secreted by the wild type of T. reesei and by the transformed strain. In particular, in FIG. 5, lane A of the isoelectric focusing gel uses partially purified CBHI from T. reesei; lane B uses wild-type T. reesei; lane C is cbh1
Lane D uses protein from a T.reesei strain in which the cbh1 and cbh2 genes have been deleted. In FIG. 5, the right side of the figure is marked to indicate the location of a single protein found in one or more secreted proteins. In particular, BG refers to β-glucosidase, E1 refers to endoglucanase I, E2 refers to endoglucanase II, and E3 refers to endoglucanase III.
C1 refers to exocellobiohydrolase I,
And C2 refers to exocellobiohydrolase II.

Figure 6A is a diagram showing the T. reesei cbh2 locus cloned as a 4.1 kb EcoRI fragment on genomic DNA.
FIG. 2 is a view showing a cbh2 gene deletion vector pPΔCBHII.

FIG. 7 shows T.reesei transformed with EcoRI cut pPΔCBHII after Southern analysis using ³² P-labeled pPΔCBHII as a probe.
FIG. 4 is an autoradiograph of DNA from strain P37PΔCBHIPyr26. The size of the molecular weight markers is shown in kilobase pairs on the left side of the figure.

 FIG. 8 is a diagram of the plasmid pEGIpyr4.

FIG. 9 shows Trichoderma reesei over a pH range at 40 ° C.
Acidic EG-rich fungal cellulase composition derived from
RBB-CMC activity profile (deleted I and II) and Trichoderma reesei over pH range at 40 ° C
FIG. 4 illustrates the activity profile of the abundant EG III cellulase composition derived from E. coli.

FIG. 10 shows a launderome of a cotton-containing fabric treated with a cellulase composition having varying amounts of CBH components.
It is a figure explaining the result of the strength loss after three washings during ter).

FIG. 11 illustrates fiber removal results (based on panel test scores) of cotton-containing fabrics treated with cellulase secreted by wild-type Trichoderma reesei at various pHs.

Figure 12 shows cotton-containing fabrics treated with various concentrations of cellulase (ppm) secreted by wild-type Trichoderma reesei and secreted by strains of Trichoderma reesei that were genetically engineered to be unable to secrete CBH I and CBH II. FIG. 7 is a view for explaining the result (based on a panel test score) of fiber removal of a cotton fabric treated with the treated cellulase.

FIG. 13 illustrates a panel test of the flexibility of EG-rich cellulase compositions at various concentrations (ppm) derived from Trichoderma reesei strains that have been genetically altered to not secrete CBH I and CBH II. is there.

FIG. 14 is a diagram of a site-specific alteration made in egl1 and cbh1 to generate convenient restriction endonuclease cleavage sites. In each case, the upper line shows the original DNA sequence, the changes introduced are shown in the middle line, and the new sequence is shown in the lower line.

FIG. 15 is a diagram of a larger EcoRI fragment obtained from pCEPC1.

FIG. 16 is an autoradiograph of DNA from an unconverted strain of T.reesei RutC30 and from two transformants obtained by converting T.reesei with EcoRI cut pCEPC1. The DNA was cut with Pst I to obtain a complementary DNA strand and hybridized with ³² P-labeled pUC4K :: cbh1. Marker DNA
Fragment sizes are shown in kilobase pairs on the left side of the figure.

FIG. 17 is a diagram of plasmid pEGII :: P-1.

FIG. 18 shows pEGII :: P- cut with HindIII and BamHI.
1 is an autoradiograph of DNA from T. reesei strain P37PΔΔ67P1 converted in 1. Prepare a complementary DNA strand,
Is about 4 of radiolabeled T. reesei DNA containing egl3 gene.
Hybridized with the kb Pst I fragment. Lines A, C and E
Contains DNA from the unconverted strain, while B, D and F
Contains DNA from an unconverted T.reesei strain. T.reese
The iDNA was ligated with Bgl II of lines A and B and EcoR of lines C and D.
Cut at V and at PstO on lines E and F. marker
DNA fragment sizes are shown in kilobase pairs on the left side of the figure.

FIG. 19 is a diagram of the plasmid pPΔEGI-1.

FIG. 20 is an autoradiograph of a complementary DNA strand of DNA isolated from a transformant of strain GC69 obtained with Hind III-cut pΔEGIpyr-3. Lane C shows the pattern of hybridization expected from the unconverted strain with the probe and radiolabeled pΔEGIpyr-3. Lane A shows the expected pattern of the transformed strain in which the eq1 gene was split, and lane B shows pΔEGIpyr
A transformed strain in which -3 DNA was integrated into the genome but did not divide the eql gene is shown. Lane D is p cut with Hind III.
Contains ΔEGIpyr-3 to provide an appropriately sized marker. The size of the marker DNA fragment is shown in kilobase pairs on the left side of the figure.

DETAILED DESCRIPTION OF THE INVENTION As mentioned above, the method of the present invention is an improvement over conventional methods of treating cotton-containing fabrics with cellulase. The improvement consists in using a specific cellulase composition that gives the fabric the desired enhanced quality, but minimizes the loss of strength of the fabric. However, before describing the present invention in detail, the following terms will first be defined.

The term "cotton-containing fabric" refers to sewn or unsewn fabrics made from pure cotton or cotton blends, including cotton knits, cotton denim, cotton strands, and the like. If a cotton blend is used, the amount of cotton in the fabric should be at least about 40
It should be weight percent cotton, preferably about 60 weight percent cotton, most preferably about 75 weight percent cotton. When used as a blend, other materials used in the fabric include polyamide fibers (eg, nylon 6 and nylon 66), acrylic fibers (eg, polyacrylonitrile fibers), and polyester fibers (eg, polyethylene terephthalate), polyvinyl It may include one or more non-cotton fibers including synthetic fibers such as alcohol fibers (eg, vinylon), polyvinyl chloride fibers, polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers, and aramid fibers. It is contemplated that regenerated cellulose, such as rayon, can be used as an alternative to cotton in the method of the present invention.

As used herein, the term "finishing" refers to applying a sufficient amount of a finish to a cotton-containing fabric to substantially hinder the cellulose hydrolyzing activity of cellulase on the fabric. Finishes generally include, for example, softness,
For the purpose of enhancing the properties of the fabric, such as drapability, it is applied at or near the end of the manufacturing process of the fabric, thereby protecting the fabric from reaction with cellulase. Useful finishes for finishing cotton-containing fabrics are known in the prior art and include melamine, glyoxal, or urea formaldehyde, as well as wax, silicon, fluorescent species, and quaternari.
es). When so finished, the cotton-containing fabric is substantially less reactive to cellulase.

The term "fungal cellulase" refers to a fungal source or an enzyme composition derived from a microorganism that has been genetically altered to contain and represent all or part of the cellulase gene obtained from the fungus. Fungal cellulases act on cellulose and its derivatives, hydrolyze cellulose and give the main products glucose and cellobiose. Fungal cellulases are distinguished from cellulases produced from non-fungal sources including microorganisms such as actinomycetes, gliding bacteria (myxobacteria) and eubacteria. Fungi capable of producing cellulases that are convenient for preparing the cellulase compositions described herein are disclosed in British Patent No. 2,094,826,
That disclosure is used herein as a reference.

The best fungal cellulases are generally acidic or neutral
Although having the greatest activity in the pH range, some fungal cellulases are known to have significant activity under neutral and slightly alkaline conditions, i.e., cellulases derived from, for example, Humicola insolens are neutral It is known to have significant activity under slightly alkaline conditions.

Fungal cellulases are known to consist of several classes of enzymes with different substrate specificities, enzymatic patterns of action, and the like. In addition, the enzyme components within each class may exhibit different molecular weights, different degrees of glycosylation, different isoelectric points, different substrate specificities, and the like. For example, fungal cellulases
Includes cellulase classes including endoglucanase (EG), exocellobiohydrolase (CBH), β-glucosidase (BG), and the like. On the other hand, although bacterial cellulases are reported in the literature to contain little or no CBH component, it is rare that CBH-like components derived from bacterial cellulases have been reported to have exocellobiohydrolase activity.

One or more CBs produced by naturally occurring fungal sources
A fungal cellulase composition consisting of H and EG components is found at the rate at which each of these components was produced by a fungal source, sometimes referred to herein as a `` complete fungal cellulase system '' or `` complete fungal cellulase composition, '' The composition may be obtained from the classification and components of cellulases isolated therefrom, from incomplete cellulase compositions produced by bacteria and certain fungi, or from cellulase CBH and / or EG.
Distinguish from cellulase compositions obtained from microorganisms that have been genetically modified to produce no or over- or under-producing components.

Fermentation methods for culturing fungi for the production of cellulases are known per se in the prior art. For example, cellulase systems are batch, fed-batch (fed-b
atch) and either a solid or submerged medium, including a continuous flow step. The accumulation and purification of the cellulase system from the fermented broth is also achieved by methods known per se in the prior art.

"Endoglucanase (" EG ") type components" are all those fungal cellulase components or Trichoderma reesei
2 shows a combination of components that exhibit fabric activity properties similar to the endoglucanase component of the present invention. In this regard, Trichoderma ree
sei endoglucanase components (especially EGI, EGI
I, EG III, etc., alone or in combination) is a cotton-containing fabric (compared to the untreated fabric) when these components are contained in a fabric treatment medium and the fabric is treated with this medium. To provide improved feel, improved appearance, softness, enhanced color, and / or stone wash appearance. In addition, the cotton-containing fabric Trichoderma reesei
The treatment with the endoglucanase component of this method results in less strength loss as compared to treatment with a similar composition but additionally containing a CBH type I component.

Thus, endoglucanase-type components provide an improved feel to cotton-containing fabrics (compared to untreated fabrics) when these components are included in the medium used to treat the fabrics, Provides an improved appearance, softness, enhanced color, and / or stone wash appearance, and the strength produced by treatment with a similar cellulase composition but additionally containing a CBH type I component. A fungal cellulase component that provides less strength loss compared to loss.

Such endoglucanase-type components can be used to (a) hydrolyze soluble cellulose derivatives such as carboxymethylcellulose (CMC), thereby producing a CM containing solution.
C, which reduces the viscosity of C; (b) readily hydrolyzes hydrated forms of cellulose such as phosphate-swollen cellulose (eg, Walces cellulose); and higher crystalline forms of cellulose (eg, Avicel, It does not include components that are classically classified as endoglucanases using activity tests, such as the ability of the components to readily, but not hydrolyze, solucaflok. On the other hand, it is believed that not all endoglucanase components, as defined by such activity tests, give cotton-containing fabrics one or more enhanced qualities, as well as reduced strength loss. . Therefore, the endoglucanase-type component is
It is more accurate for the present invention to define a component of a fungal cellulase that possesses similar fabric activity properties as owned by the endoglucanase component of derma reesei.

Fungal cellulases may contain more than one EG-type component. The different components generally have different isoelectric points, different molecular weights, different degrees of glycosylation, different substrate specificities, different patterns of enzyme activity, and the like. Separation by ion exchange chromatography or the like is enabled by the isoelectric points of different components. In fact, the isolation of components from different fungal sources is known in the prior art. See, for example, U.S. Patent Application 07 / 422,814 to Bork et al., International Application WO 89/0925 to Schlein et al.
9. Wood et al., Biochemistry and genetics of cellulose degradation, 3
1-52 (1988); Wood et al., Hydrocarbon Research, 190
279-297 (1988); Schlein, Methods in Enzymology, 160, 234-242 (1988) and the like. The full disclosure of each of these references is cited herein.

It is generally believed that a combination of EG-type components provides a synergistic response in providing enhanced quality to the cotton-containing fabric as well as providing reduced strength loss compared to a single EG component. . On the one hand, a single EG-type component is more stable and has a broad spectrum of activity over the pH range. Therefore,
The EG-type component used in the present invention is either a single EG-type component or a combination of two or more EG-type components. When a combination of components is used, the EG-type components are derived from the same or different fungal sources.

 EG-type components can be derived from bacterially derived cellulases.

"Exo-cellobiohydrolase-type (" CBH-type ") components" are CBH I and / or Trichoderma reesei.
1 shows a fungal cellulase component that exhibits fabric activity properties similar to CBH II cellulase. In this regard, Trichoderma reesei CBH I and CBH II when used in the absence of the EG type component cellulase component (as defined above)
The ingredients alone do not give the treated cotton-containing fabric a noticeable increase in feel, appearance, color and / or stonewash appearance. Therefore, when used in combination with EG-type components, Trichoderma reesei CBH I
The component provides the cotton-containing fabric with increased strength loss.

Thus, the CBH type I and CBH type II components
Trichoderma reesei CBH I and CBH II respectively
A fungal cellulase component that exhibits similar fabric activity properties to the component. As mentioned above, for CBH type I components, this includes the property of increasing the strength loss of cotton-containing fabrics when used in the presence of EG type components. In a preferred embodiment, when brought in combination with an EG-type component, Tr
The CBH type I component of ichoderma reesei provides increased cleaning effectiveness. Therefore, CBH I of Trichoderma reesei
The mold component provides an increased softening effect when used in combination with the EG type component or alone.

Such exo-cellobiohydrolase-type components are indispensable for Trichoderma reesei CBH I and CBH I
It does not include components that were classically classified as exo-cellobiohydrolases using activity tests as used to characterize I. For example, such components include (a)
Competitively inhibited by cellobiose (Ki is about 1m
M); (b) Substituted celluloses such as carboxymethylcellulose cannot be hydrolyzed significantly; (c) Hydrolysates phosphoric acid-swollen cellulose and highly crystalline cellulose does not hydrolyze much. On the other hand, the activity test
Certain fungal cellulase components, characterized as CBH components, when used alone in cellulase compositions, provide improved feel, appearance, softness, color, and / or cotton-containing fabrics with minimal strength loss. Or give a stone wash look. Therefore, defining such an exo-cellobiohydrolase as an EG-type component, because the exo-cellobiohydrolase component possesses similar functional properties for textile applications as that owned by the endoglucanase component of Trichoderma reesei Is believed to be more accurate with respect to the present invention.

For a detergent component containing a cellulase composition that is CBHI deficient, CBHI-rich or EGIII-rich, it is the amount of cellulase that produces reducing sugars from cellulose, and not the relative rate of hydrolysis of a particular enzyme component. Have been found to impart desirable cleaning properties to cotton-containing fabrics, for example, to provide the cleaning composition with one or more improved color restoration, improved softness, and improved detergency. Was done.

A fungal cellulase having one or more EG-type components and one or more CBH I-type components, wherein the fungal cellulase has a protein weight ratio of all EG-type components to all CBH I-type components of greater than 5: 1. Is obtained by a purification technique. That is, a complete cellulase system can be purified to substantially pure components by recognized separation techniques well documented in the literature, including ion exchange chromatography at appropriate pH, affinity chromatography, size exclusion, etc. . For example, in ion exchange chromatography (usually anion exchange chromatography), a pH gradient,
Alternatively, the cellulase component can be separated by elution with a salt gradient or a pH and salt gradient. After purification
The required amount of the desired component can be recombined.

It is also contemplated that a mixture of cellulase components having the required ratio of EG type to CBH type I cellulase components can be prepared by methods other than isolation and component recombination.
In this regard, the emission conditions of natural microorganisms can be polarized to provide a relatively high ratio of the EG component to the CBH component. Similarly, to produce a mixture of cellulase compositions having a relatively high ratio of the EG type component to the CBH type component, recombinant technology employs EG to the CBH type component.
A relatively high proportion of the mold component can be changed.

In view of the foregoing, the preferred method of preparing the cellulases described herein is by genetically modifying a microorganism to over-acidify one or more acidic EG-type components. Similarly, the microorganism can be genetically modified such that the method cannot produce one or more CBH-type components that do not produce any heterologous proteins. In such cases, the required amount of cellulase produced by the improved microorganism will be one or more EG type components and one or more
To provide a fungal cellulase having a CBH Type I component, wherein the fungal cellulase has a weight ratio of all EG type components to all CBH Type I components of greater than 5: 1. (Containing type I components).

In this regard, for example, US patent application Ser.
07 / 593,919 discloses a method for genetically purifying Trichoderma reesei such that it cannot produce one or more CBH components and / or cannot overexpress one or more EG components. In addition, the method of use is a Trichoderma reese that does not represent any heterologous protein.
Produce i strains. Similarly, Miller et al., "Asperqillus
"Direct and non-direct gene replacement in nidulans" Molecules and Cellular Biology, 1714-1721 (1985) describe Asperqillus nidula by DNA-mediated transformation using linear fragments of heterologous DNA.
Disclosed are methods for deleting genes in ns. Miller et al. Achieve gene deletion without producing any heterologous proteins.

CBH type I and / or CBH
Deletion of the gene responsible for producing the type II cellulase component has the effect of enriching the amount of type EG component present in the cellulase composition.

Fungal cellulase compositions are contemplated for use herein from fungal sources that produce low levels of CBH type I components.

In addition, one or more CBHs produced by conventional methods
The required amount of the type I component is a cellulase composition produced from a microorganism that has been genetically manufactured such that the CBH type I component cannot be produced so as to achieve a specific ratio of the EG type component to the CBH type I component. Added. That is, a cellulase composition that is free of all CBH-type components so as to be enriched in EG-type components can be achieved simply by adding 2 weight percent of the purified CBH type I component (or CBH II component) to the cellulase composition 2% by weight of CBH I
It is formulated to contain a mold component (or a CBH type II component).

“Β-Glucosidase (BG) component” refers to a cellulase component that exhibits BG activity, ie, such component acts from the non-reducing end of cellobiose and other cellooligosaccharides (“cellobiose”) and as a single product Give glucose. The BG component does not adsorb or react on the cellulose polymer. In addition, such BG components are competitively inhibited by glucose (Ki is about 1 mM). In a strict sense, BG components are not literally cellulases because they cannot degrade cellulose, but such BG components
The components are defined as cellulase systems because these enzymes promote the overall degradation of cellulose by further degrading the inhibitory cellulose degradation products (particularly cellobiose) produced by the binding action of the CBH and EG components. Contained within. In the absence of the BG component, the hydrolysis of crystalline cellulose occurs only slowly or hardly. The BG component is often p-nitrophenol BD-
It is characterized by an allyl substrate such as glucoside (PNPG) and is therefore often referred to as allyl-glucosidase. Not all allyl glucosidases are BG components, some do not hydrolyze cellobiose.

The presence or absence of the BG component in the cellulase composition is believed to be used to modulate the activity of the CBH component in the composition (ie, a non-CBH type I component). That is, because cellobiose is produced during cellulase degradation by the CBH component, and since high concentrations of cellobiose are known to inhibit CBH activity, such cellobiose is further hydrolyzed to glucose by the BG component Therefore, the absence of the BG component in the cellulase composition "disappears" the CBH activity when the cellobiose concentration reaches the inhibition level
Let it. Also, one or more additives (eg, cellobiose, glucose, etc.) may be added directly or indirectly to some or all of the CB to the cellulase composition.
It is believed that type I activity, as well as other CBH activities, can be effectively "eliminated". If such an additive is used, the amount of additive used may be reduced to a level equal to or less than the level of CBH type I activity achieved by using the cellulase compositions described herein.
The resulting composition is considered to be a composition suitable for use in the present invention if it is sufficient to reduce type I activity.

On the other hand, if the level of cellobiose produced by the CBH component limits such overall hydrolysis in the absence of the added BG component, then the cellulase composition containing the added amount of the BG component will have a cellulosic content of cellulose. Increases overall hydrolysis.

Any method of increasing or decreasing the amount of the BG component in the cellulase composition is described in Office Specification No. 01
0055-056, filed December 10, 1990, entitled "Saccharification of Cellulose by Cloning and TRICHODERMA RE
No. 07 / 625,140, entitled "Amplification of β-Glucosidase Gene by ESEI," which is hereby incorporated by reference in its entirety.

Fungal cellulases can contain one or more BG components. The different components generally have different isoelectric points that allow their separation, such as by ion exchange chromatography. Either a single BG component or a combination of BG components is used.

When used in fabric treatment solutions, the BG component is generally added in an amount sufficient to prevent cellulase inhibition of any CBH and EG components found in the cellulase composition. The amount of BG component added depends on the amount of cellobiose produced in the fabric composition, which is easily determined by one skilled in the art. However, when used, B compared to any CBH-type components present in the cellulase composition
The weight percent of the G component is preferably from about 0.2 to about 10 weight percent, more preferably from about 0.5 to about 5 weight percent.

Preferred fungal cellulases used to prepare the fungal cellulase compositions used in the present invention are Trichoderm
a reesei, Trichoderma koningii, Pencillum, sp.,
Humicola insolens, etc. Certain fungal cellulases are commercially available. Cellcast (obtained from NovoIndustry, Copenhagen, Denmark), rapidase (obtained from Gistbrocade, Delft, WV, The Netherlands), cytolase 123 (South San Francisco, CA,
Obtained from Zinenka International). Other fungal cellulases can also be easily isolated by well-known fermentation and isolation methods.

The term "buffer solution" refers to well-known acid / base reagents that stabilize cellulase solutions to undesirable pHs that fluctuate during cellulase treatment of cotton-containing fabrics. In this regard, cellulase activity is known to be pH dependent. That is, a particular cellulase composition exhibits a cellulose hydrolyzing activity within a defined pH range, and optimal cellulose hydrolyzing activity is generally found within a defined small portion thereof. The specific pH range of cellulose hydrolyzing activity will vary with each cellulase composition. As mentioned above, most cellulases are:
Some cellulase compositions exhibit activity of hydrolyzing cellulose in an acidic to neutral pH profile, but exhibit activity of hydrolyzing cellulose in an alkaline pH profile.

During cellulase treatment of cotton-containing fabrics, the pH of the initial cellulase solution may be outside the range required for cellulase activity. Further, during processing of the cotton-containing fabric, the pH may change, for example, due to the formation of reaction products that change the pH of the solution. In any case, the pH of the non-interfering cellulase solution can be outside the range required for the activity of hydrolyzing cellulose. When this occurs, an undesirable reduction or cessation of the activity of hydrolyzing cellulose in the cellulase solution occurs. For example, if a cellulase with a production activity profile is used in an aqueous solution that is not neutrally buffered, the pH of the solution
Will result in a lowering of the cellulose hydrolyzing activity and possibly of the cellulose hydrolyzing activity. On the other hand, the use of cellulases having a neutral or alkaline profile in an aqueous solution that is not neutrally buffered initially provides significant cellulose hydrolysis activity.

In view of the above, the pH of the cellulase solution should be kept within the range required for the activity of hydrolyzing cellulose. One means of accomplishing this is by simply monitoring the pH of the system and adjusting the desired pH by addition of either an acid or a base. However, in a preferred embodiment, the pH of the system is preferably kept within the desired pH range by using a buffer in the cellulase solution. Generally, in order to maintain the pH of the solution within a range in which the cellulase used exhibits activity,
Use a sufficient amount of buffer. The particular buffer used will be selected in relation to the particular cellulase composition used, as long as the different cellulase compositions have different pH ranges that exhibit cellulose hydrolyzing activity. The buffer chosen for use in the cellulase used can be readily determined by one skilled in the art, taking into account the pH range and the optimal conditions for the cellulase used and the pH of the cellulase solution. Preferably, the buffer used is one that is compatible with the cellulase composition and maintains the pH of the cellulase solution within the pH range required for optimal activity. Suitable buffers include sodium citrate, ammonium acetate, sodium acetate, disodium phosphate, and other buffers known in the art.

The tensile strength of a cotton-containing fabric can be measured with respect to the vertical and transverse directions that are perpendicular to each other. Thus, as used herein, the term "vertical tensile strength" refers to the tensile strength of a cotton-containing fabric measured along the length of the cotton-containing fabric, while the term "lateral tensile strength" Refers to the tensile strength of the cotton-containing fabric measured across the width of the cotton-containing fabric. The tensile strength of the cotton-containing fabric treated with the cellulase solution is compared with the tensile strength before treatment with the cellulase solution to determine the strength-reducing effect of the treatment. If the tensile strength is reduced too much, the resulting cotton-containing fabric easily tears and / or forms pores. Therefore, it is desirable to maintain a tensile strength after treatment that is at least about 50% of the tensile strength before treatment (both vertical and horizontal).

The tensile strength of the cotton-containing fabric is easily derived according to ASTM D1682 test methodology. Apparatus suitable for testing the tensile strength of such fabrics includes a Scott tester or an Instron tester, both of which are commercially available.
In testing the tensile strength of the cotton-containing fabric treated with the cellulase solution, care must be taken to avoid shrinkage of the fabric after treatment and before testing. Such shrinkage causes incorrect tensile strength data.

Enhanced quality for cotton-containing fabrics is achieved by the methods used herein. For example, cotton-containing fabrics having improved feel are disclosed in Japanese Patent Laid-Open Nos. 58-3617 and 58-5.
4032 and Oishi et al., "Reforming cotton fabric with cellulase" and JTN December 1988 journal article, "New, weight loss treatment to soften the feel of cotton fabric."
Can be achieved by The teachings of each of these documents are hereby incorporated by reference.

Similarly, a method of improving both the feel and appearance of cotton-containing fabrics is to add the aqueous solution containing cellulase under conditions such that the solution is agitated and the cascade effect of the cellulase solution is achieved across the cotton-containing fabric. Including contacting the fabric. Such a method leads to an improved feel and appearance of the cotton-containing fabric so treated, and
No. 07 / 598,506, filed October 16, 1990, which is hereby incorporated by reference in its entirety.

A method of enhancing cotton-containing knits is described in the second edition of the 1990 International Spinning Bulletin, page 5, dyeing / printing / finishing, which is hereby incorporated by reference.

Similarly, a method of imparting the appearance of a stonewash to cotton-containing denim is described in US Pat. No. 4,832,864, which is hereby incorporated by reference in its entirety.

Other methods of enhancing cotton-containing fabrics by treatment with a cellulase composition are also known in the prior art. Preferably, in such a method, the treatment of the cotton-containing fabric with cellulase is performed before finishing the cotton-containing fabric.

As noted above, the invention is an improvement over prior art methods of treating cotton-containing fabrics, as long as the invention uses a specific cellulase composition that minimizes the strength loss of the treated fabric. The cellulase composition used herein is a fungal cellulase composition comprising one or more EG-type components and one or more CBH-type components, wherein the cellulase composition comprises
It has a weight ratio of all EG type components to all CBH type components of greater than 5: 1.

In addition, the use of the cellulase compositions described herein provides enhanced fabric / color in stressed cotton-containing fabrics. That is, during the manufacture of the cotton-containing fabric, the fabric is stressed, and if so stressed, the fabric contains broken and disturbed fibers. Such fibers detrimentally give the fabric an even worn appearance. However, when treated in accordance with the method of the present invention, the fabric so stressed will have an enhanced fabric / color. This is likely to be caused by removing some of the broken and disturbed fibers that have the effect of restoring the appearance of the fabric before stress is applied.

In addition, by using the cellulase compositions described herein for pigmented dyed fabrics (eg, denim), these cellulase compositions cause less redeposition of dye. Also, their anti-redeposition properties are:
It is believed that one or more specific EG-type components are enhanced compared to other components.

The fungal cellulase composition described above is used in an aqueous solution containing cellulase and other optional components including, for example, buffers, surfactants, scouring agents, and the like. The concentration of cellulase used in this solution is generally sufficient for its intended purpose. That is, the cellulase composition is used in an amount that provides the desired enhanced quality to the cotton-containing fabric. The amount of cellulase composition used can also be
Equipment used, parameters of the method used (temperature of cellulase solution, exposure time to cellulase solution, etc.),
Cellulase activity (eg, a cellulase solution requires a lower concentration for a more active cellulase composition as compared to a less active cellulase composition), and the like. The exact concentration of the cellulase composition can be readily determined by one skilled in the art based on the above-mentioned factors as well as the desired effect.
Preferably, as used herein, the concentration of the cellulase composition in the cellulase solution ranges from 0.01 grams / liter of the cellulase solution to about 10.0 grams / liter of the cellulase solution;
More preferably, from 0.05 grams / liter of the cellulase solution to about 2 grams / liter of the cellulase solution. (The cellulase concentration mentioned above refers to the weight of total protein).

When a buffer is used in the cellulase solution, the concentration of the buffer in the aqueous cellulase solution is sufficient to maintain the pH of the solution within a range where the cellulase used exhibits an activity depending on the characteristics of the cellulase used. It is. The exact concentration of buffer used will depend on a number of factors that can be readily considered by one skilled in the art. For example, in a preferred embodiment, the buffer and the buffer concentration are selected to maintain the pH of the cellulase solution within the pH range required for optimal cellulase activity. Generally, the buffer concentration in the cellulase solution is about 0.005N or greater. Preferably, the concentration of the buffer in the cellulase solution is from about 0.01 to about 0.5N, more preferably from about 0.05 to about 0.15N. It is also possible that the increased buffer concentration in the cellulase solution increases the rate of loss of tensile strength of the treated fabric.

In addition to the cellulase and buffer, the cellulase solution can optionally contain small amounts, ie, less than about 2 weight percent, and preferably about 0.01 to about 2 weight percent of a surfactant. Suitable surfactants include, for example, textiles, including anionic, nonionic and amphoteric surfactants, and any surfactant that is compatible with cellulase.

Suitable anionic surfactants for use herein are linear or branched alkyl benzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl or alkenyl groups; alkyl or alkenyl sulfates Olefin sulfonates; alkanesulfonates and the like. Suitable counterions for anionic surfactants are alkali metal ions such as sodium and potassium; alkaline earth metals such as calcium and magnesium; ammonium ions;
Or an alkanolamine having three to three alkanol groups.

The ampholyte surfactant includes a quaternary ammonium salt sulfonate, a betaine-type ampholyte surfactant and the like. Such ampholyte surfactants have both positively and negatively charged groups in the same molecule.

The nonionic surfactant generally comprises a polyoxyalkylene ether, a higher fatty acid alkanolamide or an alkylene oxide adduct thereof, a fatty acid glycerin monoester, and the like.

 Mixtures of such surfactants are also used.

As used herein, the solution ratio, i.e., the weight ratio of cellulase solution to fabric, is generally in an amount sufficient to achieve the desired enhanced quality of the cotton-containing fabric, and the method used and achieved. Depends on quality. Preferably, the solution ratio is generally about 0.1: 1 or greater, more preferably greater than about 1: 1 and even more preferably about 10: 1.
Greater than 1. Using a solution ratio of greater than about 50: 1 is usually not preferred from an economic point of view.

The reaction temperature for cellulase treatment is determined by two competing factors. First, higher temperatures generally correspond to increased reaction kinetics, i.e., faster reactions, which result in reduced reaction times compared to the reaction times required at lower temperatures. Thus, the reaction temperature is generally at least about 30 ° C. or higher. Second, cellulases are proteins that lose activity above a predetermined reaction temperature, the temperature of which depends on the properties of the cellulase used. Therefore, if the reaction temperature becomes too high, the activity of hydrolyzing cellulose is lost as a result of denaturation of cellulase. As a result, the maximum reaction temperature used herein is generally about 65 ° C. In view of the above, the reaction temperature is generally from about 30 ° C to about 65 ° C, preferably from about 35 ° C to about 60 ° C, more preferably from about 35 ° C to about 50 ° C.

Reaction times are generally from about 0.1 hours to about 24 hours, preferably from about 0.25 hours to about 5 hours.

Cotton-containing fabrics treated in the manner described above using such a cellulase composition have a reduced strength loss compared to the same cotton-containing fabric treated in a similar manner with the complete fungal cellulase composition.

In a preferred embodiment, a concentrate can be prepared for use in the method described above. Such concentrates preferably contain concentrated amounts of the above-described cellulase compositions, buffers and detergents in aqueous solution.
When so formulated, the concentrate can be easily diluted with water to quickly and accurately prepare a cellulase solution having the requisite concentration for these additives. Preferably, such a concentrate comprises about 0.1 to about 20 weight percent of the above-described cellulase composition (protein); about 10 to about 50 weight percent buffer; about 10 to about 50 weight percent surfactant; And about 0 to 80 weight percent water. When aqueous concentrates are formulated, these concentrates are diluted by a factor of about 2 to about 200 to reach the required concentration of the components in the cellulase solution. As is readily apparent, such a concentrate allows for easy dispensing of the cellulase solution, as well as convenient transfer of the concentrate to the point of use. The above-mentioned cellulase composition is added to a concentrate in any state such as a liquid diluent, a granular form, an emulsion, a gel, and a paste. Such forms are well-known to those skilled in the art.

When a solid cellulase concentrate is used, the cellulase composition is generally granular, powdered, aggregated, and the like. If granules are used, the granules can preferably be formulated to include a cellulase protecting agent. For example, as an office specification number 010055-073, January 17, 1991
See U.S. patent application Ser. No. 07 / 642,669, filed on Jan. 10, entitled "Granules Containing Both Enzyme and Enzyme Protectant, and Detergent Compositions Containing Such Granules". This application is hereby incorporated by reference in its entirety. Similarly, the granules can be formulated to contain substances that reduce the rate of dissolution of the granules in the cleaning medium. Such materials are available from Office Statement No.GCS-171-US
No. 07 / 642,596, filed Jan. 17, 1991 and entitled "Particulate Composition,"
The application is included here in its entirety.

The cellulase compositions described herein may also be used in pre-washing and be used as a pre-soak, either liquid or spray. Further, it is contemplated that the cellulase compositions described herein may also be used at home as one composition suitable for enhanced color and textile appearance. See, for example, U.S. Patent No. 4,738,682. It is hereby incorporated by reference in its entirety.

The following examples are provided to illustrate the invention and should not be construed as limiting its scope.

Examples Examples 1-12 and 22-30 demonstrate that Tric genetically engineered to be unable to produce one or more cellulase compositions or to overproduce certain cellulase compositions.
Explain the production of hoderma reesei.

Example 1 Selection of a pyr4 derivative of Trichoderma reesei The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for uridine biosynthesis. The toxicity inhibitor 5-fluoroorotic acid (FOA) is contained in uridine by wild-type cells and therefore poisons the cells. However, cells detected in the pyr4 gene are resistant to this inhibitor, but require uridine for growth. therefore,
It is possible to select a pyr4 derivative strain using FOA. Indeed, spores of T. reesei strain RL-P37 (Seal-Nice, G. and Montencoat, BS, Appl. Microbiol. B
iotechnol. 20, p. 46-53 (1984)) was spread on the surface of a solidified medium containing 2 mg / ml uridine and 1.2 mg / ml FOA. Within 3 or 4 days, natural FOA-resistant colonies emerge, after which those FOA-resistant derivatives that require uridine for growth can be identified. To identify those derivatives that had a specifically defective pyr4 gene, protoplasts were produced and transformed with a plasmid containing the wild-type pyr4 gene (see Examples 3 and 4). Following conversion, protoplasts were spread on media lacking uridine. Subsequent growth of the transgenic colony indicated complementation of the defective pyr4 gene by the plasmid borne pyr4 gene. In this way, strain GC69 was identified as a pyr4 derivative of strain RL-P37.

Example 2 Preparation of CBHI Deletion Vector The cbh1 gene encoding the CBHI protein was hybridized with an oligonucleotide probe designed based on the published sequence for this gene using known probe synthesis methods. Genomic DNA of T.reesei strain RL-P37
(Shoemaker et al., 1983b). cbh1
The gene is present on a 6.5 kb pst I fragment and is in Pts I cut pUC4K (NJ, Piscataway, purchased from Pharmacia) by replacing the Kan ^r gene of this vector using techniques known to those skilled in the art. This technique has been described by Maniatis et al. (1989) and is hereby incorporated by reference. The resulting plasmid, pUC4K :: chb1
Was cut with Hind III and a larger fragment of about 6 kb was isolated and religated to give pUC4K :: cbh1ΔH / H (see FIG. 1). This method uses the complete cbh1 coding sequence and about 1.2 kb
Remove upstream and 1.5 kb downstream flanking sequences. Approximately 1 kb of flank DNA remains from either end of the original Pst I fragment.

The T.reesei pyr4 gene was cloned as a 6.5 kb HindIII fragment of genomic DNA in pUC18 according to the method of Maniatis et al. To form pTpyr2 (Smith et al., 1991). Plasmid pUC4K :: cbh1ΔH / H was cut with HindIII and its ends were dephosphorylated with calf intestinal alkaline phosphatase. This terminally dephosphorylated DNA contains the T.reesei pyr4 gene
Ligation with the 6.5 kb Hind III fragment yielded pΔCBHIpyr4.
FIG. 1 illustrates the construction of this plasmid.

Example 3 Isolation of protoplasts In a 500 ml flask, 100 ml of YEG (0.5% yeast extract, 2% glucose) was added to about 5 × 10 ⁷ T.reesei GC69.
Mycelium was obtained by inoculation with a spore (pyr4 derivative strain). Then, shake the flask at 37 ° C for about 16
Cultured for hours. The mycelium was harvested by centrifugation at 2,750 × g. The harvested mycelium was further washed in a 1.2 M sorbite solution and a 5 mg / ml Novozyme 234 solution (1,3-alpha-glucanase, 1,3-beta-glucanase, laminarinase, obtained from Novo Biolabs, Ct. Danbury, Trademark of a multi-component enzyme system containing xylase, chitinase and protease); 5 mg / ml M
gSO ₄ · 7H ₂ O; were resuspended in a solution of 40ml containing 1.2M sorbitol; 0.5 mg / ml of bovine serum albumin. Protoplasts were removed from cell debris by filtration through Miracloth (Calbiochem, CA, La Jolla),
Collected by centrifugation at 0 g. The protoplasts were washed three times in 1.2 M sorbite, washed once in 1.2 M sorbite, 50 mM CaCl ₂ , centrifuged,
The sorbit was resuspended at a density of about 2 × 10 ⁸ protoplasts per ml of 50 mM CaCl ₂ .

Example 4 Conversion of fungal protoplasts by pΔCBHIpyr4 200 μl of the protoplast suspension prepared in Example 3 was added to 20 μl of TE buffer (10 ml Tris, pH 7.4, 1
EcoRI cut pΔCBHIpyr4 (prepared in Example 2) in 25 mM PEG 4000, 0.6 M KCl and 50 mM
It was added to a 50 μl polyethylene glycol (PEG) solution containing mM CaCl ₂ . This mixture was incubated on ice for 20 minutes. After this culture period, 2.0 ml of PEG identified above was added thereto, the solution was further mixed and cultured at room temperature for 5 minutes. After this second culture, 4.0 ml of a solution containing 1.2 M sorbite and 50 mM CaCl ₂ was added thereto and the solution was mixed further. The protoplast solution was then added to Bogels medium N containing additional 1% glucose, 1.2 M sorbitol and 1% agarose.
(3 grams of sodium citrate per liter,
5g KH ₂ PO _4, 2 grams NH ₄ NO _3, and of 0.2 grams, MgS
_{O 4 · 7H 2 O, CaCl} 2 · 2H 2 O of 0.1 g, 5 [mu] g of α- biotin, citric acid 5mg, ZnSO ₄ · 7H ₂ O in 5 mg, 1 mg of Fe (NH
_{4) 2 · 6H 2 O,} CuSO of 0.25mg ₄ · 5H ₂ O, MnSO of 50 [mu] g ₄ · 4
Immediately added to the dissolution aliquots of H ₂ O). The protoplast / medium mixture was then poured onto a solid material containing the same bogel medium as described above. There is no uridine in the medium, and therefore only the transgenic population has pΔCBHI
of the strain GC69 by the wild-type pyr4 gene inserted into pyr4
could grow as a result of complementation of the pyr4 mutation. These colonies were subsequently transferred and 1
Purified on solid Bogel medium N containing 5% glucose and stable transformants were selected for further analysis.

At this stage, a stable transformant is defined as a transformant that is unstable due to its faster growth rate and the formation of circular colonies with smooth contours rather than jagged on solid culture media lacking uridine. Distinguished. In some cases, transformants are grown on a solid, non-selective medium (ie, containing uridine), spores are harvested from this medium, and then germinated to form a selective medium lacking uricin. Further stability tests were performed by determining the percentage of these spores that grew.

Example 5 Analysis of Transformants After transformants were grown in liquid Bogel medium N containing 1% glucose, the DNA was purified from Example 4
Was isolated from the transformant obtained in Step 1. These transformant DNA samples were further digested with Pst I restriction enzyme and subjected to agarose gel electrophoresis. The gel was then blotted onto a nitrane membrane filter and hybridized with a ³² P-labeled pΔCBHIpyr4 probe. Select the probe, 6.5kb Pst
The native cbh1 gene as an I fragment, any DNA sequence derived from the transformed DNA fragment and the native pyr4 gene were identified.

Radioactive bands from hybridization were visualized by autoradiography. The autoradiography is shown in FIG. As described above, the five samples A, B, C,
D and E were tested. E is the unconverted strain GC69 and was used as a control in this analysis. Lanes A to D show the transformants obtained by the method described above. The number on the side of the autoradiography indicates the size of the molecular weight marker. As can be seen from this autoradiography, lane D did not contain the 6.5 kb CBHI band, indicating that this gene was entirely deleted during transformation due to the incorporation of the DNA fragment at the cbh1 gene. The cbh1 deletion strain is P37PΔ
Called CBHI. Figure 2 shows one cbh1 on the T.reesei chromosome.
1 shows a schematic of the deletion of the T. reesei cbh1 gene by integration through generation of a double cross of a larger EcoR I fragment from pΔCBHIpyr4 at the locus. Other transformants analyzed would be identical to the untransferred control strain.

Example 6 Analysis of transformants harboring pIntCBHI The same procedure as in Example 5 was used in this example except that the probe used was replaced by a ³² P-labeled pIntCBHI probe. This probe is a pUC-type plasmid containing a 2 kb BglII fragment from the cbh1 locus in the region deleted in pUC4K :: cbh1ΔH / H. In this example, control, sample A which is unconverted strain GC69, transformant P3
Two samples containing 7PΔCBHI were analyzed. As can be seen from FIG. 4, as indicated by the band at 6.5 kb, sample A
It contained the bh1 gene. However, the transformant, sample B, does not contain this band at 6.5 kb and therefore does not contain the cbh1 gene and does not contain any sequences derived from the pUC plasmid.

Example 7 Protein Secretion by Strain P37PΔCBHI Produced spores from P37PΔCBHI strain were isolated from 1% glucose, 0.14% (NH ₄ ) ₂ SO ₄ , 0.2% KH ₂ PO ₄ , 0.03%
MgSO ₄ , 0.03% urea, 0.75% bactotryptone (bac
totryptone), 0.05% Tween 80, 0.00001
Of _{6% CuSO 4 · 5H 2 O} , 0.001% of _{FeSO 4 · 7H 2 O, 0.000128} %
Of _{ZnSO 4 · 7H 2 O, 0.0000054} % of _{Na 2 MoO 4 · 2H 2 O} , and
50 ml of Trichoderma containing 0.0000007% MnCl.4H ₂ O
Inoculated in basal medium. While vibrating the medium, 25
The cells were cultured in a 0 ml flask at 37 ° C. for about 48 hours. The resulting mycelium was collected by filtration through mycloth Miracloth (Calbiochem) and washed two or three times with 17 mM potassium phosphate. The mycelium was finally suspended in 17 mM potassium phosphate with 1 mM scphorose and cultured for 24 hours at 30 ° C. with further shaking. The supernatant was then collected from these cultures and the mycelium was discarded. Samples of the culture supernatant were analyzed by isoelectric focusing using a Pharmacia Fast Gel system and a pH 3-9 precast gel according to the manufacturer's instructions. The gel was stained with silver dye to visualize the protein bands. The band corresponding to the cbh1 protein was absent in samples derived from strain P37PΔCBHI, as shown in FIG. This isoelectric focusing gel shows different proteins in different supernatant cultures of T. reesei. Lane A is partially purified CBHI, lane B is supernatant from untransformed T. reesei culture, and lane C is supernatant from strain P37PΔCBHI produced by the method of the present invention. The locations of the various cellulase components are labeled with CBHI, CBHII, EGI, EGII, and EGIII. CBHI is 50% of all extracellular proteins
CBHI is the major secreted protein and therefore the darkest band on the gel. This isoelectric focusing gel clearly shows the deletion of the CBHI protein in the P37PΔCBHI strain.

Example 8 Preparation of pPΔCBHII The cbh2 gene of T. reesei encoding the CBHII protein has been cloned as a 4.1 kb EcoRI fragment of genomic DNA shown in the diagram of FIG. 6A (Chen et al., 1987, Biotechnology 5: 274-). 278). This 4.1 kb fragment was ligated with EcoRI of pUC4XL.
Inserted between sites. The latter plasmids are in the order shown here: EcoR I, BamH I, Sac I, Sma I, Hind III, Hho
I, Bal II, Cla I, Bg III, Xho I, Hind III, Sma I,
PUC derivative containing a multiple cloning site with a symmetrical pattern of restriction endoglucanase sites arranged in Sac I, BamH I, EcoR I (composed of RM Baker from Zinenker International). Using methods known in the art, plasmids, pPΔ
CBHII (Fig. 6B) is constructed, where (CBHII translation initiation site
At the Hind III site (at 74 bp 3 ') and the last codon
The 1.7 kb central region of this gene between the Cla I site (at 265 bp 3 ') was removed and the 1.6 kb containing the T.reesei pyr4 gene was removed.
b Replaced with Hind III-Cla I DNA.

The T.reesei pyr4 gene was excised from pTpyr2 (see Example 2) on a 1.6 kb NheI-SphI fragment, and SphI of pUC219 was removed.
And XbaI site (see Example 25) to produce p219M (Smith et al., 1991, Curr. Genet 19p.27-3).
3). The pyr4 gene was then added to one end with 7 bp of DNA,
It has 6 bp of DNA at the other end and was removed as a HindIII-ClaI fragment derived from the pUC219 multiple cloning site and inserted into the HindIII and ClaI sites of the cbh2 gene to form plasmid pPΔCBHII (No. 6).

Cleavage of this plasmid with EcoR I
A fragment with 0.7 kb flanking DNA from the cbh2 locus, 1.7 kb flanking DNA from the cbh2 locus at the other end and the middle T. reesei pyr4 gene is liberated.

Example 9 Deletion of the cbh2 gene in T. reesei strain GC69 According to the method outlined in Examples 3 and 4, a protoplast of the strain is produced and transformed with EcoRI cut pPΔCBHII. The DNA from the transformant is cut with EcoRI and Asp718 and agarose gel electrophoresis is performed. DN from gel
A was blotted to a membrane filter and according to the method of Example 11.
Hybridize with ³² P-labeled pPΔCBHII. Transformants with a single copy of the EcoRI fragment from pPΔCBHII integrated at exactly the cbh2 locus are identified. The transformant also
Proteins grown in shake flasks as in Example 7 and in the culture supernatant are tested by isoelectric focusing. In this way, a T. reesei GC69 transformant that does not produce CBHII protein is produced.

Example 10 Production of pyr4 derivative of P37PΔCBHI Spores of the transformant deleted for the cbh1 gene (P3
7PΔCBHI) was developed on FOA containing media. Subsequently, a pyr4 derivative of this transformant was obtained using the method of Example 1. This pyr4 strain was designated as P37PΔCBHIPyr26.

Example 11 Deletion of the cbh2 gene in a strain previously deleted for cbh1 Strain P37PΔCBHIPyr26 was produced and EcoT I-cut pPΔCBHII according to the method outlined in Examples 3 and 4.
Turned.

The purified stable transformant was cultured in a shake flask as in Example 7, and the protein in the culture supernatant was tested by isoelectric focusing. One transformant that did not produce any CBHII protein (designated P37PΔΔCBH67) was identified. Lane D of FIG. 5 shows the supernatant from a transformant lacking both the cbh1 and cbh2 genes produced according to the method of the present invention.

DNA was cut with EcoRI and Asp718 strain P37PΔΔCBH67
And subjected to agarose gel electrophoresis. DNA from this gel was blotted onto a membrane filter and labeled with ³² P-labeled pP.
Hybridization occurred with ΔCBHII (FIG. 7). Lane A of FIG.
1 shows the hybridization patterns observed for DNA from unconverted T. reesei strains. 4.1k containing wild-type cbh2 gene
b EcoRI fragment was observed. Lane B shows strain P37PΔ
1 shows the hybridization pattern observed for ΔCBH67.
One 4.1 kb band was removed and replaced by two bands of about 0.9 and 3.1 kb. This is EcoR I from pPΔCBHII
This is the pattern that would be expected if one copy of the fragment was integrated exactly at the cbh2 locus.

Identical DNA samples were also cut with EcoRI and performed as described above for Southern analysis. In this example, the probe was ³² P-labeled pIntCBHII. This plasmid contains a portion of the cbh2 gene coding sequence from within that fragment of the cbh2 gene deleted in plasmid pPΔCBHII. No hybridization was seen for DNA from strain P37PΔΔCBH67,
This indicates that the cph2 gene has been deleted and that no sequences derived from the pUC plasmid were present in this strain.

Example 12 Construction of pEGIpyr4 The T. reesei eq11 gene encoding EGI is a 4.2 kb Hind of genomic DNA from strain RL-P37 by hybridization with orthonucleotides synthesized according to the published sequence.
It was cloned as a III fragment (Pentilla et al., 1986, gene 45: 253-263; Van Earthdel et al., 1987, Bio / Technology 5: 60-64). A 3.6 kb Hind III-BamHI fragment was removed from this clone and cut with Hind III cut pU.
C218 (identical to pUC219 but with multiple cloning sites in opposite orientation, see Example 25) and pTpy
The plasmid pEGIpyr4 was obtained by ligation with a 1.6 kb HindIII-BamHI fragment containing the T.reesei pyr4 gene obtained from r2 (see Example 2) (FIG. 8). Cleavage of pEGIpyr4 with HindIII is based on T.reesei genomic DNA (egl1 and pyr4 genes)
DNA fragments excluding the 24 bp synthetic DNA between the two genes and the 6 bp synthetic DNA at one end (FIG. 8).

Example 13 Purification of cytolase 123 cellulase into cellulase component Cytase 123 cellulase was fractionated as follows. The usual distribution of the cellulase component of this cellulase system is as follows.

CBH I 45-55% by weight CBH II 13-15% by weight EGI 11-13% by weight EG II 8-10% by weight EG III 1-4% by weight BG 0.5-1% by weight The fraction contains the following resins Sephadex G-25 gel filtration resin from Sigma Chemical Company (MO, St. Louis), SP Trisacryl M cation exchange resin and QA Trisacryl M anion from IBF Biotechnics (MD, Sabaji). Exchange resin. pH6.8
10 mM sodium phosphate buffer and Sephadex G-25
0.5 g of cytolase 123 cellulase was desalted using a 3 liter column filled with gel filtration resin. The desalted solution was then loaded onto a 20 ml column of QA Trisacryl M anion exchange resin. The fraction bound to this column is CBHI
And EGI. These components can be used from 0 to about 50
Separated by gradient elution using an aqueous gradient containing 0 mM sodium chloride. Fractions that did not bind to this column contained CBH II and EG II. These fractions are p
Desalting was performed using a column of Sephadex G-25 gel filtration resin equilibrated with 10 mM sodium citrate at H3.3.
200 ml of this solution was then loaded onto a 20 ml column of SP Trisacryl M cation exchange resin. CBH II and EG
II was eluted separately using an aqueous gradient containing 0 to about 200 mM sodium chloride.

Other cellulase systems that can be separated into their components according to methods analogous to those of Example 13 described above include Cellcast (obtained from NovoIndustry, Copenhagen, Denmark), Lapitase (from Gystprocade, NV, Delft, NV, Netherlands). Obtained), and Tr
Includes cellulase systems derived from ichoderma koningii, Penicillum sp.

Example 14 Purification of EG III from cytolase 123 cellulase Example 13 described above showed the isolation of several components from cytolase 123 cellulase. However, EG
Since III was not present in very small amounts in cytolase 123 cellulase, the following method was used to isolate this component.

A. Large Scale Extraction of EG III Cellulase Enzyme 100 liters of cell-free cellulase filtrate was heated to about 30 ° C. The heated material has about 4% weight / volume PEG 800
It was prepared from 0 (molecular weight of about 8000, polyethylene glycol) and about 10% weight / volume anhydrous sodium sulfate. The mixture formed a two-phase liquid mixture. The phases were separated using a SA-1 disc stack centrifuge. The phases were analyzed using a silver stained isoelectric focusing gel. Separation provided EG III and xylanase.
The removed composition contains about 20 to 50 weight percent EG
III.

For the method described above, the use of polyethylene glycol having a molecular weight of less than about 8000 is unsuitable for separation,
On the other hand, the use of polyethylene glycol having a molecular weight greater than about 8000 will eliminate the desired enzyme in the withdrawn composition. With respect to the amount of sodium sulfate, levels of sodium sulfate greater than 10% weight / volume cause precipitation problems, while levels of sodium sulfate less than 10% weight / volume result in insufficient separation or solution remaining in one phase. became.

B. Purification of EG III by Fractionation EG III was purified from the complete fungal cellulase composition produced by the wild-type Trichoderma reesei (CA, Genencor, South San Francisco, Cytase 123 cellulase, commercially available from International). Fractionation is performed using the following resins: Sephadex G-25 gel filtration resin from Sigma Chemical Company (Mo, St. Louis), SP Trisacryl M from IBF Biotechnics (MD, Sabaji). Performed using a column containing cation exchange resin and QA Trisacryl M anion exchange resin using a 3 liter column filled with 10 mM sodium phosphate buffer pH 6.8 and Sephadex G-25 gel filtration resin. Then 0.5 g of cytolase 123 cellulase was desalted, and the desalted solution was then washed with QA Trisacryl M anion exchange resin It was loaded onto a column of 20 ml. The fraction bound on this column contained CBH I and EG I. Fractions which do not bind to this column, CBH II, EG I
Includes I and EG III. These fractions were converted to 10 mM
Sephadex G- equilibrated with sodium citrate
Desalting was performed using a column of 25 gel filtration resin. Then, 200 ml of this solution was mixed with 2 parts of SP Trisacryl M cation exchange resin.
Loaded into a 0 ml column. EG III was eluted with 100 ml of 200 mM sodium chloride.

To increase the isolation efficiency of EG III, one or more EG
Trichoderma reesei genetically modified to be unable to produce I, EG II, CBH I and / or CBH II
It is desirable to use The absence of one or more such components necessarily leads to a more efficient isolation of EG III.

Similarly, the EG III composition described above is further purified,
It is desirable to provide a substantially pure EG III composition, ie, a composition containing greater than about 80 weight percent of the protein. For example, such substantially pure EG III protein is obtained by using the material obtained from Method A in Method B, or by using the material obtained from Method B in Method A. One particular method of further purifying EG III is by further fractionation of the EG III sample obtained in part b) of this Example 14. Further fractionation was performed using a Mono-S-HR 5/5 column (obtained from NJ, Piscataway, Pharmacia LKB Biotechnology).
Performed by the PLC system. The FPLC system consists of a liquid chromatography controller, two pumps, a dual passage monitor, a fraction accumulator and a chart recorder (all obtained from NJ, Viscataway, Pharmacia LKB Biotechnology). Fractionation was performed as in Example 14.
5 ml of the EG III sample prepared in part b) of
This was done by desalting on a 20 ml Sephadex G-25 column equilibrated with 10 mM sodium citrate.
The column was then eluted with a 0-200 mM aqueous gradient of NaCl at a rate of 0.5 ml / min against Sample I collected in a 1 ml fraction. EG III was removed in fractions 10 and 11 and determined by SDS gel electrophoresis to be more than 90% pure. This pure EG III is suitable for determining the N-terminal amino acid sequence by known techniques.

Substantially pure EG purified in Example 13 above
The III and EG I and EG II components are used alone or in a mixture in the method of the invention. These EG components have the following properties: MW pI Optimum pH ¹ EG I about 47-49 kD 4.7 about 5 EG II about 35 kD 5.5 about 5 EG III about 25-28 kD 7.4 about 5.5-6.0 1. Examples below Optimal pH measured by RBB-CMC activity by 15.

The use of these mixtures in the practice of the present invention results in a synergistic response that improves softness, feel, appearance, etc. as compared to a single component. On the other hand, the use of a single component in the practice of the present invention results in a more stable or broad spectrum of activity over the pH range. For example, Example 15 below shows that EG III is RBB-CM under alkaline conditions.
Shows significant activity against C.

Example 15 Activity of Cellulase Composition over a pH Range The following method was used to determine the pH profile of two different cellulase compositions. The first cellulase composition comprises CBH I prepared from Trichoderma reesei genetically modified in a manner similar to that described above so that it cannot produce CBH I and CBH II components and
It was a II deletion cellulase composition. The cellulase composition generally comprises a cellulase composition derived from Trichoderma reesei, from about 58 to 70 percent CBH.
As long as it does not contain I and CBH II, this cellulase composition is essentially free of CBH type I and CBH type II cellulase components, and thus the EG component,
I, EG II, EG III, etc. are abundant.

The second cellulase composition was a pure fraction of EG III, about 20 to 40%, isolated from a cellulase composition derived from Trichoderma reesei by a purification method similar to part b) of Example 14. .

The activity of these cellulase compositions was measured at 40 ° C,
The measurement was performed using the following method.

Add the appropriate enzyme solution, 5 to 20μ, at a concentration sufficient to provide the required amount of enzyme in the final solution. pH 4,
5, 5.5, 6, 6.5, 7, 7.5 and 8, 2 weight percent RBB-CMC in 0.05M citrate / phosphate buffer
250 μm (Remazol brilliant blue R-carboxymethylcellulose, commercially available from Megazyme, NSW2151, North Rock, 6 Altona Place, Australia).

Shake and incubate at 40 ° C for 30 minutes. Cool in an ice bath for 5 to 10 minutes. Add 1000 μl of methyl cellosolve containing 0.3 M sodium acetate and 0.02 M zinc acetate. Stir and leave for 5-10 minutes. Centrifuge and pour the supernatant into the cuvette. 590n of solution in each cuvette
Measure the optical density (OD) at m. Higher levels of optical density correspond to higher levels of enzyme activity.

The results of this analysis are set forth in FIG.
2 illustrates the relative activities of CBH I and II deleted cellulase compositions as compared to cellulase compositions. From this figure,
Cellulase compositions lacking CBH I and CBH II have optimal cellulose hydrolyzing activity on RBB-CMC around pH 5.5 and have an alkaline pH, i.e.
It has some activity at a pH of 8 above 7. On the other hand,
The EG III-rich cellulase composition has optimal cellulose hydrolyzing activity at pH 5.5-6 and alkaline pH.
Has considerable activity.

From the above examples, those skilled in the art need only adjust and maintain the pH of the aqueous fabric composition so that the cellulase composition is active, preferably having optimal activity. As noted above, such adjustment and retention involves the use of appropriate buffers.

Example 16 Cellulase Composition Landalometer Strength Loss Assay This example tests the ability of a cellulase composition to reduce the strength of a cotton-containing fabric. Trichode
This example shows that the activity of most cellulase compositions derived from rma reesei is maximal at or around pH 5 and thus the intensity loss is most pronounced when the assay is performed around this pH. Use an aqueous cellulase solution maintained at pH5.

That is, in this example, the first cellulase composition analyzed was a complete fungal cellulase system produced from wild-type Trichoderma reesei (CA, a cytolase 123 cellulase commercially available from Zinenker International, South San Francisco). And is identified as GCO10.

The second cellulase composition analyzed was from 1 to 12 above and from 22 below so that CBH II could not be expressed.
Tricho genetically modified in a manner similar to the 30 examples
CBH II deleted cellulase composition prepared from derma reesei and identified as CBHIId. As long as CBH II comprises up to about 15 percent of the cellulase composition, deletion of this component will be at abundant levels of CBH I, and all EG components.

The third cellulase composition analyzed was CBH I and C
A CBH I and CBH II deficient cellulase composition prepared from Trichoderma reesei that has been genetically modified in a manner similar to that described above so that BH II cannot be expressed, and is identified as CHBI / IId. Unless CBH I and CBH II are produced by this improved microorganism, the cellulase will not necessarily contain all CBH type I components, as well as CBH components.

The last cellulase composition analyzed was CBH I prepared from Trichoderma reesei genetically modified in a manner similar to that described above so that CBH I could not be expressed.
Deletion cellulase composition, identified as CBHId. As long as the improved microorganism does not produce CBH I, the cellulase composition does not necessarily contain all CBH type I components.

The cellulase compositions described above were tested for the effect of the strength loss of the cotton-containing fabric in the Landometer. The composition was first standardized so that equal amounts of the EG component were used. Add each cellulase composition and titrate to pH 5,
400 ml of a solution of 20 mM citrate / phosphate buffer containing 0.5 ml of nonionic surfactant was separated. Each purified solution was added to a separate Landalometer canister.
In these canisters, there is an amount of marble that promotes the loss of strength, as well as 16 inches x 20 inches (about 40.6 cm x about 40.6 cm).
50.8 cm) of cotton fabric (NJ08846, Middlesex, 200 Blackford Street, available as Style 467 from Test Fabric). The canister was then closed and lowered into a landrometer maintained at 43 ° C. The canister was then spun in the bath at a speed of at least 40 revolutions per minute (rpm) for about one hour. Thereafter, the clothes were taken out, rinsed well, and dried in a standard dryer.

To maximize the strength loss results, the above method was repeated two more times, and after the third treatment, the cotton fabric was removed and analyzed for strength loss. The strength loss was determined by the tensile strength in the transverse direction using an Instron tester ("FT
S ") was measured and the results were compared to the FTS of a fabric treated with the same solution except that no cellulase was added. The results of this analysis are reported as percent strength loss determined by the following formula: The results of this analysis are shown in FIG. 10, which shows that CBH I,
That is, compositions containing total cellulase (GC010) and CBH II deficient cellulase have the greatest loss of strength, while compositions containing no CBH I have significantly more loss compared to total cellulase and CBH II deficient cellulase. It shows that it has a reduced strength loss. These results indicated that the presence of the CBH type I component in the cellulase composition provided the composition with increased strength loss as compared to a similar composition that did not contain the CBH type I component.

Similarly, these results indicate that CBH II plays a role in intensity loss.

Thus, in view of these results, the strength loss resistant cellulase composition is a composition that does not include all CBH type I cellulase components, and preferably all CBH type cellulase components. In this regard, it is believed that such cellulase compositions result in lower strength loss at pH ≧ 7 than their results observed at pH 5 shown in FIG.

During the manufacture of the cotton-containing fabric, the fabric may be stressed, and when so stressed, the fabric contains broken and disturbed fibers. Such fibers give the fabric an even worn appearance. However, it has been found that the method of the present invention results in increased fabric / color. It is believed that this is done by removing broken and disturbed fibers that have the effect of restoring the appearance of the fabric before stress is applied.

Examples 17 and 18 below illustrate this advantage of the present invention. It is noted that these examples used worn cotton T-shirts (knits) as well as new cotton knits. The faded appearance of a worn cotton-containing fabric is caused by the accumulation of broken fibers and open-surface fibers of the previous period on the fabric. These fibers give the fabric a faded and tangled appearance, and thus removal of these fibers is a necessary requirement to restore the fabric to its original vivid color. In addition, the accumulation of broken surface fibers on new cotton knits gives such fabrics a unique appearance. Thus, these experiments are necessarily applicable to enhancing the color of stressed cotton-containing fabrics, both of which involve the removal of surface fibers from the fabric.

Example 17 Color Enhancement The ability of the EG component to enhance color in cotton-containing fabrics was analyzed in the following experiments. That is, the first experiment was performed at various pH
Wild-type Tric removes surface fibers from a wide range of cotton-containing fabrics
A complete cellulase system produced by hoderma reesei (C
A. Measure the ability of Cytase 123, commercially available from Jinencar International, South San Francisco. The cellulase was tested for its ability to remove surface fibers in a landrometer. In the final composition
An appropriate amount of cellulase, providing either 25 ppm or 100 ppm cellulase, was added to a 400 ml separation solution of 20 mM citrate / phosphate buffer containing 0.5 mg nonionic surfactant. Samples were prepared and titrated to provide samples at pH 5, pH 6, pH 7, and pH 7.5. Each of the prepared solutions was then added to a separate Landalometer canister. In these canisters, an amount of marble that promotes fiber removal, as well as 7 inches by 5 inches (about 17.8 inches)
cm × about 12.7cm) cotton fabric (NJ08846, middle sex,
Blackford Street 200, 100% woven cotton, available as Style 439W from Test Fabric). The canister was then closed and lowered into a landrometer maintained at 43 ° C. The canister is then turned on at a speed of at least about 40 revolutions per minute (rpm).
Rolled in the bath for hours. The clothes are then removed, rinsed well and dried in a standard dryer.

The fabric so treated was then analyzed for fiber removal by panel test evaluation. In particular, the fabric (not marked) was evaluated for fiber level by six individual. The fabric was evaluated visually for surface fibers and on a scale of 0 to 6. This scale has six standards for making meaningful comparisons. The standards are: Evaluation standard ^a 0 Fabric not treated with cellulase 1 Fabric ^b treated with 8 ppm cellulase 2 Fabric treated with 16 ppm cellulase 3 Fabric treated with 20 ppm cellulase 4 Fabric treated with 40 ppm cellulase 5 50 ppm 6) Fabric treated with 100 ppm cellulase a) In all standards, the fabric was 100% cotton sheet standardized test fabric (Style 439W) from NJ08846, Middlesex, Blackford Street 200, Test Fabric. Number).

b) All samples were treated with the same cellulase composition. Cellulase concentrate was total protein. The conditions of the landrometer treatment are shown in Example 16 described above.

The fabric to be evaluated was given the most closely matched rating of the standard. After a complete analysis of the fabric, the values given to each fabric by all of the individuals were added and the average value was obtained.

The result of this analysis is shown in FIG. Thus, FIG. 11 shows that at the same pH, a dose-dependent response is seen as the amount of fiber removed. That is, at the same pH,
Fiber treated with more cellulase provided a higher level of fiber removal compared to fiber treated with less cellulase. Furthermore, the results of this form show that at higher pH, fiber removal is still affected by simply using a higher concentration of cellulase.

Second, in an experiment, two different cellulase compositions were compared for their ability to remove fibers. That is, the first cellulase analyzed was wild-type Trichoderma reese
A complete cellulase system (Cytolase 123, commercially available from Zinencar International, South San Francisco, CA) produced by i and is termed GCO10.

The second cellulase composition analyzed was CBH
Tirchoderma reese genetically modified in a manner similar to that described above so that it cannot express I and CBH II
a cellulase composition substantially free of all CBH type components (including CBH type I components) prepared by i.
Identified as an HI / II deletion. As long as CBH I and CBH II make up up to about 70 percent of the cellulase composition, deletion of this component will be at abundant levels of all EG components.

These compositions were tested for their ability to remove surface fibers in a landrometer. An appropriate amount of cellulase to provide the required concentration of the EG component in the final composition,
It was added to a 400 ml separation solution of 20 mM citrate / phosphate buffer containing 0.5 mg of nonionic surfactant. Samples were prepared and titrated to pH5. Each of the prepared solutions was then added to a landrometer canister. In these canisters, a certain amount of marble that promotes fiber removal,
A 7 inch x 5 inch (about 17.8 cm x about 12.7 cm) cotton fabric (NJ08846, Middlesex, 200 Blackford Street, 100% woven cotton, available as Style 439W from Test Fabric) was added. The canister was then closed and lowered into a landrometer maintained at 43 ° C. The canister was then spun in the bath at a speed of at least about 40 revolutions per minute (rpm) for 1 hour. The clothes are then removed, rinsed well and dried in a standard dryer.

The fabric thus treated was then analyzed for fiber removal by the panel test described above. The results of this analysis are shown in FIG. 12, plotted based on estimated EG concentrations. Thus, FIG. 12 shows that the GC010 and CBH I / II deletion cellulase compositions gave substantially the same fiber removal results at substantially equal EG concentrations. This form of the results suggests that it is the EG component that provides the fiber removal. These results, in conjunction with the results of FIG. 11, indicate that the EG component removes surface fibers.

Example 18 Tergotometer Color Enhancement This example further relates to Example 17,
This example demonstrates that the H-type component is not required for color enhancement and the purpose of this example is to investigate the ability of cellulase compositions lacking the CBH type component to enhance color on cotton-containing fabrics. It is in.

That is, the cellulase composition used in this example was CBH
Trichode genetically modified in a manner similar to that described above so that I and CBH II cannot be expressed
As prepared from rma reesei, this composition is substantially free of all CBH type components (containing CBH type I components). CBH I and CBH II form about the cellulase composition.
To the extent that it constitutes up to 70 percent, deletion of this component results in abundant levels of all EG components.

The assay was performed by adding a sufficient concentration of this cellulase composition to 50 mM citrate / phosphate buffer to provide 500 ppm of cellulase. The solution was titrated to pH 5 and 0.1 weight percent nonionic surfactant (NC273)
60, commercially available from Thomasville, Gresco Nfg.). 10 inches x 10
Inch (about 25.4cm x about 25.4cm) faded cotton-containing fabric,
As well as 10 inches x 10 with loosely broken surface fibers
New knitted fabric of inch (about 25.4cm x about 25.4cm)
Dispose in 1 liter of this buffer, and add 110F ゜ (about 43.3
C) for 30 minutes, and then stirred for 30 minutes at at least 100 revolutions per minute. The fabric was then removed from the buffer, washed and dried. The resulting fabric was then compared to the fabric before treatment. The results of this analysis are shown below: cotton-containing material Worn cotton T-shirt Profitable New cotton knit Profitable Broken containing surface fibers resulting from use of tergotometer The term "profited", including the removal of surface fibers, indicates the restoration of color (i.e., does not fade) of the treated fabric as compared to the untreated fabric. These results demonstrate the results of Example 17 that the presence of the CBH-type component is not required to effect color restoration of the faded cotton-containing fabric.

Since the use of such cellulase compositions removes broken / loose fibers created during processing without harmful loss of strength to the fabric, the use of the compositions would provide benefits during fiber processing. Conceivable.

Example 19 Softness This example demonstrates that the presence of a CBH type component is not required to provide improved softness to a cotton-containing fabric. That is,
This example includes all CBH-type components whose composition was derived from Trichoderma reesei, which was genetically produced by the method described above such that it was unable to produce CBH I and CBH II components. Use no cellulase composition.

The cellulase composition was tested for its ability to soften a terry wash cloth. That is, 14 inches x 15
Inches (about 35.6cm x about 38.1cm), not flexible
8.5 oz (about 241 g) cotton terry woven cloth (obtained as NJ08846, Middlesex, 200 Blackford Street, Style 420NS from Test Fabric)
It was cut into 7 inch × 7.5 inch (about 17.8 cm × about 19.1 cm) pieces.

The cellulase compositions described above were tested for their ability to soften these shards in a landrometer.
That is, 500 ppm, 250 ppm, 10 ppm in the final cellulase composition.
Appropriate amounts of CBH I and II deletion cellulases are provided at 0.0, 50, and 10 ppm to provide 0, 50, and 10 ppm of cellulases.
25 weight percent nonionic surfactant (Triton X11
4) was added to a 400 ml separation solution of 20 mM citrate / phosphate buffer. In addition, containing the same solution,
A blank without cellulase was made. The sample so prepared was titrated to pH5. Each of the prepared solutions was then added to a separate Landalometer canister. In these canisters, an amount of marble to promote softness was added, as well as the cotton snips described above. All conditions were performed in triplicate with two snips per canister. The canister was then closed and lowered into a landrometer maintained at 37 ° C. The canister was then spun in the bath at a speed of at least about 40 revolutions per minute (rpm) for 1 hour. The clothes were then removed, rinsed well and dried in a standard dryer.

The pieces were then tested for flexibility by evaluation in a selection test. In other words, six panelists,
Each individual was given a series of swatches and instructed to evaluate the swatches for softness based on softness criteria such as overall fabric softness. Pieces and blanks resulting from treatment with five different enzyme concentrations were placed behind the screen and the panelists were instructed to order the least soft to the softest. Each piece was scored based on its order relative to the other pieces; 5 was the softest and 0 was the least soft. Scores from each panelist were accumulated and averaged.

The result of this averaging is shown in FIG. That is, these results show that higher concentrations provide improved flexibility. It can be seen that this improved softening is achieved without the presence of either CBH I or II in the cellulase composition.

Example 20 Feeling and Appearance This example illustrates that the presence of a CBH type component is not essential to provide an improved feel and appearance to a cotton-containing fabric. Thus, this example demonstrates that Trichoderma reesei has been genetically engineered in the manner described above so that it cannot produce any CBH type components (ie, it cannot produce CBH I and II components). The derived cellulase composition is used.

The cellulase composition was tested for its ability to improve the appearance of the cotton-containing fabric. That is, in the appearance of this embodiment, a 100% cotton sheet of appropriate size (NJ0884
6, available as Middlesex, 200 Blackford Street, Style 439W from Test Fabric)
Was used.

The cellulase compositions described above were tested for their ability to improve the appearance of these samples in a landrometer.
That is, 25 ppm, 50 ppm, and
Appropriate amounts of CBH I and II to provide 100 ppm of cellulase
The deleted cellulase was reduced to 20 mM citric acid / 0.025% by weight containing a nonionic detergent (Triton X114).
The phosphate buffer was added to 400 ml of the separation solution. In addition, a blank was made containing the same solution but without any cellulase. The sample so prepared was titrated to pH5. Each of the prepared solutions was then added to a separate landrometer canister. Then close the canister, 37
Lowered in a landerometer maintained at ° C. The canister is then turned at least about 40 revolutions per minute (rp
m) in the bath for 1 hour. The clothes were then removed, rinsed well and dried in a standard dryer.

The samples were then analyzed for improved appearance by evaluation of the selection test. That is, six panelists were given four samples (not showing discrimination) and asked to evaluate their appearance. For panelists, the term "appearance"
Refers to the physical appearance of a cotton-containing fabric to the eye, and is determined in part by the presence or absence of fuzz, surface fibers, etc., on the surface of the fabric, and by the ability or inability to discern the structure (weave) of the fabric. Was instructed. Woven fabrics with little fuzz and surface fibres, whose structure (weave) is clearly discernable, have an improved appearance compared to fabrics with fuzzy and / or loose fibers and / or indistinguishable weaves .

The panelists then scored each sample based on their order, comparing to the other samples: 4 is the best appearance and 1 is the worst appearance. Scores from each panelist were aggregated and averaged. The results of this test are shown below.

Cellulase Amount Average Appearance None 125 ppm 2 50 ppm 3 100 ppm 4 The CBH I and II deleted cellulase compositions were then tested for their ability to improve the feel of cotton-containing fabrics. That is, in the appearance of this embodiment, 100
% Cotton sheet (NJ08846, Middlesex, Blackford Street 200, Style 439 from Test Fabric Inc.
(Obtained as number W) was used.

The cellulase compositions described above were tested for their ability to improve the appearance of these samples in a landrometer.
That is, an appropriate amount of cellulase providing 500 ppm, 1000 ppm, and 2000 ppm of cellulase in the final cellulase solution was added to 24 L of a 20 mM citrate / phosphate buffer. In addition, a blank was made containing the same solution but without any cellulase. All tests were performed at pH 5.8 and performed in an industrial washing machine. The washer was operated at 50 ° C., 24 L total volume, 50: 1 (weight to weight) liquid to fabric ratio, and the machine was operated for 30 minutes. Thereafter, the sample was removed and dried in an industrial dryer.

The samples were then analyzed for improved feel by evaluation of the selection test. That is, five panelists were given four samples (not showing discrimination) and asked to evaluate for feel. To panelists, fabrics with improved feel are smoother and silkier than other fabrics,
To distinguish that feel from quality, such as softness (meaning the softness of the fabric rather than its feel), thickness, color, or other physical properties not included in the smoothness of the fabric. Supported.

The panelists then scored each sample based on their order, comparing to the other samples: 4 is the best feel and 1 is the worst feel. Scores from each panelist were aggregated and averaged. The results of this test are shown below.

Cellulase Amount Average Appearance None 1.5 ± 0.5 500 ppm 1.7 ± 0.4 1000 ppm 3.2 ± 0.4 2000 ppm 3.8 ± 0.4 The above results show that an improvement in feel and appearance can be achieved with a cellulase composition that does not include all CBH-type components.

Example 21 Stone Wash Appearance This example demonstrates that the presence of a CBH type component is not essential to give a cotton containing fabric a stone wash appearance. In other words, this example shows that any CBH
Also cannot produce the type component (ie, CBH I
Trichoderma reese genetically produced by the method as described above so that it cannot produce
Cellulase composition derived from i, and Trichoderma
A complete cellulase composition derived from reesei and obtained as Sidelase 123 from Zinenker International, South San Francisco, California is used.

These cellulase compositions were tested for their ability to give a stonewashed appearance to denim pants containing dyed cotton. That is, samples were prepared using an industrial washing machine and dryer under the following conditions: 10 mM citrate / phosphate buffer at pH 5 Total volume 40 L 110 F ゜ (about 43.3 ° C.) 4 sets of denim pants 1 hour operation Samples were evaluated for the appearance of the stone wash by eight panelists with 50 ppm CBH I and II deleted cellulase or 100 ppm complete cellulase (ie, approximately equal to EG concentration). All eight panelists select 100 ppm total cellulase over non-enzymatic treated pants as having a good stonewash appearance. Four out of eight panelists chose CBH I and II deleted cellulase-treated pants over all cellulases as having a good stonewash appearance, while the other four panelists Select all cellulase treated pants as having a stone wash appearance. These results indicate that CBH I and I
I-deleted cellulase-treated pants are indistinguishable from all cellulase-treated pants, and CBH I and / or CBH II
Indicates that the cotton-containing fabric is not desperate to give the stonewash the appearance.

For Examples 16 to 21, no CBH type I component, T
Cellulase compositions derived from microorganisms other than richoderma reesei could be used in place of the cellulase compositions described in these examples. In particular, the source of the cellulase composition containing the EG-type component is not critical to the invention and any fungal cellulase composition containing one or more EG-type components and substantially free of all CBH I-type components Things are used here. For example, fungal cellulases used to prepare the fungal cellulase compositions used in the present invention include Trichoderma koningii, Pencill
Cellulase obtained from um sp. or the like or commercially available is used, that is, cell cast (obtained from Novo Industry, Copenhagen, Denmark), rapidase (obtained from Gistbrocade, Delft, Netherlands), and the like. . In Example 16, CB
The cotton-containing fabric was treated with the cellulase composition containing HI and / or CBH II. In the fungal cellulase composition not containing CBH I, all the EG-type components
Experiments were performed with the addition of the CBH type I component in an amount greater than 5: 1 with respect to the type component, and satisfactory results were obtained.

Example 22 Conversion of Trichoderma reesei Containing Plasmid pEGIpyr4 According to the method outlined in Example 1, T. reesei strain Ru
pyr4 deficient derivative of tC30 (Seal Nice and Munteng Coat, (1984), Appl. Microbiol. Biotechnol. 20 :: 46-
53). Protoplasts of this strain are transformed into uncut pEGI
After transformation with pyr4, stable transformants were purified.

Five of these transformants (EP2, EP4, EP5, EP6, EP
11), inoculated with unconverted RutC30 in 50 ml of YEG medium in 250 ml shake flask at 28 ° C. for 2 days
The cells were cultured with shaking. The resulting mycelium was washed with sterile water, and 50 ml of TSF medium (0.05 M citrate-phosphate buffer, pH 5.0; Avicel microcrystalline cellulose, 10 g / l; KH
_{2 PO 4, 2.0g / l;} (NH 4) 2 SO 4, 1.4g / l; proteose peptone, 1.0 g / l; _{urea, 0.3g / l; MgSO 4 ·} 7H 2 O, 0.3g / l; CaCl
_{2, 0.3g / l; FeSO 4} · 7H 2 O, 5.0mg / l; MnSO 4 · H 2 O, 1.6mg /
l; ZnSO ₄ , 1.4 mg / l; CoCl ₂ , 2.0 mg / l; 0.1% Tween 80)
Added. These cultures were further cultured with shaking at 28 ° C. for 4 days. Supernatant samples were taken from these cultures and assayed to determine the total amount of protein and endoglucanase activity as described below.

The endoglucanase assay was performed using Remazol Brilliant Blue Carboxymethyl Cellulose (Australia,
RBB-CM obtained from NSW, North Rock, Megazyme
This is done by releasing the eluted staining oligosaccharides from C). The medium was prepared by adding 2 g of dry RBB-CMC to 80 ml of vigorously stirred and freshly boiled deionized water. When cooled to room temperature, 5 ml of 2M sodium acetate buffer (p
H4.8) was added to adjust the pH to 4.5. The volume was finally adjusted to 100 ml with deionized water and sodium azide was added to a final concentration of 0.02%. Aliquots of T. reesei control cultures, pEGIpyr4 transformant culture supernatants or 0.1 M sodium acetate (10-20μ) as blanks were placed in tubes, 250μ medium was added and the tubes were added to 37 μl.
Incubated at 30 ° C for 30 minutes. Place the tube on ice for 10 minutes, 1 ml
Of cold precipitant (3.3% sodium acetate, 0.4% zinc acetate, pH5 with HCl, 76% ethanol) was added. The tube was vortexed and left for 5 minutes before centrifugation at approximately 13,000 xg for 3 minutes. Optical density was measured spectrophotometrically at a wavelength of 590-600 nm.

The protein assay used was a BCA (Bicinchonic Acid) assay using reagents obtained from Pierce, Rockford, Illinois, USA. The standard was calf serum albumin (BSA). BCA reagent is combined with one part of reagent B
Prepared by mixing with 50 parts of reagent A. 1 ml of BCA reagent
Mix with 50 μl of appropriately diluted BSA or reagent culture supernatant. The culture was carried out at 37 ° C. for 30 minutes, and the optical density was finally measured photometrically at a wavelength of 562 nm.

Table 1 shows the test results described above. It is clear that some of the produced transformants have increased endoglucanase activity compared to the untransformed strain RutC30. Exo-cellobiohydrolases and endoglucanases produced by unconverted T. reesei appear to make up 70 and 20 percent, respectively, of the total amount of secreted protein. Therefore, transformants such as EP5, which produce almost four times more endoglucanase than strain RutC30, are expected to secrete approximately equal amounts of endoglucanase-type and exo-cellobiohydrolase-type proteins.

The transformants described in this example were obtained using the complete pEGIpyr4 and contain the DNA sequence integrated into the genome derived from the pUC plasmid. Before the conversion, Hind
It is possible to cut pEGIpyr4 with III and isolate a larger DNA fragment containing only T.reesei DNA. T.ree
Transformation of sei with this isolated fragment of DNA allows the isolation of transformants that overproduce EGI and do not contain any heterologous DNA sequences except for the two short pieces of synthetic DNA shown in FIG. Also, using pEGIpyr4, cbh1 gene, or cbh2
It is also possible to convert strains in which the gene, or both genes, have been deleted. In this way, strains are constructed that overproduce EGI, produce a limited range of exo-cellobiohydrolases, or produce no exo-cellobiohydrolases.

The method of Example 22 is used to produce a T. reesei strain that overproduces any of the other cellulase compositions, xylanase components or other proteins normally produced by T. reesei.

The results described above are provided for the purpose of indicating the overproduction of the EGI component relative to the total protein, and not for indicating the degree of overproduction. In this regard, overproduction will vary for each example.

Example 23 Construction of pCEPC1 A plasmid, pCEPC1, was constructed. Here, the EGI coding sequence was functionally fused to a promoter from the cbh1 gene. This is 5 '(upstream) of each translation start site.
The DNA sequence of the cbh1 and egl1 genes to create a convenient restriction endonuclease cleavage site.
It is performed using in vitro site-specific sudden induction. DNA
Sequence analysis was performed to confirm the expected sequence at the junction between the two DNA fragments. Diagram of specific changes made
See Figure 14.

The DNA fragment ligated to form pCEPC1 is pUC4K
Of the cbh1 locus, a 2.1 kb fragment from the 5 'flank region. It contains the promoter region, extends into the manufactured Bcl I site,
Does not include the bh1 coding sequence.

B) Starting less than 5 'with the BamHI site created,
A 1.9 kb fragment of genomic DNA from the egl1 locus containing approximately 0.5 kb beyond the translation stop codon. PUC2 at the 3 'end of the fragment
There are 18 bp derived from 18 multiple cloning sites, 15
A bp synthetic oligonucleotide is used to ligate this fragment with the following fragment.

C) From about 1 kb downstream of the cbh1 translation stop codon
A fragment of DNA from the 3 'flank region of the cbh1 locus, extending 2.5 kb downstream. D) 3.1 kb of DNA containing the T. reesei pyr4 gene obtained from pTpyr2 (Example 2) and having 24 bp of DNA at one end derived from the pUC18 multiple cloning site
The NheO-SphI fragment was inserted into the NheI site of fragment (C).

The plasmid, pCEPC1, was constructed by incorporating the EGI coding sequence at the cbh1 locus and introducing CET without introducing any heterologous DNA into the host strain.
It was designed to replace the BHI coding sequence. Cleavage of this plasmid with EcoR I, cbh1 promoter region, eg
a fragment containing the l1 coding sequence and transcription termination region, T.reesei
from the 3 '(downstream) flank region of the pyr4 gene and the cbh1 locus
Exfoliate the DNA fragment (see FIG. 15).

Example 24 Transformant Containing pCEPC1 DNA pyr4 deficient strain of T. reesei RutC30 (Seal-Nice,
Was obtained by the method outlined in Example 1.
This strain was transformed by EcoRI cut pCEPC1. Stable transformants were selected and subsequently cultured in shake flasks for cellulase production as described in Example 22. To visualize the cellulase protein, electrophoresis of an isoelectric focusing gel was performed on samples from these cultures using the method described in Example 7. Of a total of 23 transformants analyzed by this method, 12
It turns out that it does not produce any HI protein,
Expected results of integration of pCEPC1 DNA at the h1 locus.
Using Southern analysis, integration occurred completely at the cbh1 locus in some of these transformants and the absence of any sequence derived from the bacterial plasmid vector (pUC4K) (see FIG. 16). ). For this analysis, DNA from transformants was cut with PstI before electrophoresis and blotting on membrane filters. The generated Southern blot was labeled with the radiolabeled plasmid pUC4.
Probed with K :: cbh1 (Example 2). The probe hybridized to the cbh1 gene on a 6.5 kb fragment of DNA from an unconverted control culture (FIG. 16, lane A). DN at cbh1
Integration of the pCEPC1 fragment of A appears to result in the loss of this 6.5 kb band and three other bands corresponding to the approximately 1.0 kb, 2.0 kb and 3.0 kb DNA fragments. This is the correct pattern observed in the transformant shown in FIG. 16, lane C.
As shown in FIG. 16, lane B is an example of a transformant in which multiple copies of pCEPC1 were integrated at a site in the genome other than the cbh1 locus.

An endoglucanase activity assay was performed as in the untransformed culture, and the sample was diluted 50-fold prior to the assay, such that the protein concentration in the sample was between about 0.03 and 0.07 mg / ml. Performed on samples of culture supernatant from transformants, as described in 22. Unconverted control culture and 4
Two different transformants (CEPC1-101; CEPC1-103, CEPC
Table 2 shows the results of the tests performed on the test samples (referred to as 1-105 and CEPC1-112). Transformants CEPC1-103 and CEPC1-1
12 is an example in which integration of the CEPC1 fragment resulted in loss of CBHI production.

The results described above are provided for the purpose of indicating the overproduction of the EGI component relative to the total protein, and not for indicating the degree of overproduction. In this regard, overproduction will vary for each example.

Other T.re similar to pCEPC1, but replacing the egl1 gene
It is possible to construct a plasmid having the esei gene. In this way, overexpression of other genes and cb
Simultaneous deletion of h1 gene could be achieved.

In addition, other genes such as T.re in which cbh2 has been deleted in advance.
It is also possible to construct a transformant which does not produce any exo-cellobiohydrolase and overexpresses endoglucanase, for example, by converting the esei pyr4 derivative strain with pCEPC1.

A construct similar to pCEPC1 is used, but when replacing DNA from another locus in T.reesei with DNA from the cbh1 locus, T.reesei
It is also possible to insert the gene under the control of another promoter at another locus in the genome.

Example 25 Construction of pEGII :: P-1 The egl3 gene (previously called EGIII by others) encoding EGII was cloned from T. reesei and its DN
The A sequence has been published (Saroheimo et al., 1988, gene 63:11).
-21). From strain RL-P37, the PstI site and Xh of pUC219
o About 4 kb of genomic DNA inserted between I sites Pst I-Xh
o The gene was obtained as an I fragment. The vector pUC219 is Bgl I
Derived from pUC219 by extending the multiple cloning site to include the I, Cla I and Xho I restriction sites (gene 77: 6, described by Wilson et al. In 1989).
9-78). Using methods known in the art, T. reese present on a 2.7 kb Sal I fragment of genomic DNA
The i pyr4 gene was inserted into the SalI site within the EGII coding sequence, producing plasmid pEGII :: P-1 (FIG. 17). This resulted in fragmentation of the EGII coding sequence without any sequence deletion. The plasmid, pEGII :: P-1, was cut with HindIII and BamHI, both of which were derived from the multiple cloning site of pUC219, 5 bp above one end and 16 bp at the other end. Linear fragments of DNA exclusively derived from reesei can be produced.

Example 26 T. reesei GC for producing a strain that cannot produce EGII
Transformation of 69 with pEGII :: P-1 T. reesei strain GC69 is transformed with pEGII :: P-1 previously cut with HindIII and BamHI, and stable transformants are selected. Total DNA was isolated from those transformants and Southern analysis was used to identify these transformants, where the DN containing the pyr4 and egl3 genes
The fragment of A was integrated at the egl3 locus, resulting in fragmentation of the EGII coding sequence. The transformant cannot produce EGII. Alternatively, a strain in which any or all of the cbh1, cbh2, or egl1 gene has been deleted can be converted using pEGII :: P-1. Thus, a strain that produced a certain cellulase component and did not produce any EGII component could be constructed.

Example 27 Conversion of T. reesei with pEGII :: P-1 to Produce Strains Incapable of Producing CBHI, CBHII, and EGII According to the method outlined in Example 1, strain P37PΔΔCB
A pyr4-deficient derivative of H67 (from Example 11) was obtained. This strain P37ΔΔ67P1 was transformed with pEGII :: P-1 previously cut with HindIII and BamHI, and stable transformants were selected. Total DNA was isolated from those transformants and Southern analysis was used to identify these transformants, where a fragment of the DNA containing the pyr4 and egl3 genes was integrated at the egl3 locus, As a result, the EGII coding sequence was fragmented. The Southern blot described in FIG.
Egl cloned into the Pst I site of
Probed with an approximately 4 kb Pst I fragment of T. reesei DNA containing the three genes. When DNA isolated from strain P37ΔΔ67P1 was digested with PstI for Southern analysis, the egl3 locus was subsequently visualized as a mono-4 kb band on an autoradiograph (FIG. 18, lane E). However, for transformants in which the egl3 gene had been disrupted, this band was lost and replaced by two new bands as expected (FIG. 18, lane F). When the DNA was cut with EcoR V or Bgl II, the size of the band corresponding to the egl3 gene was unconverted P37Δ
About 2.7 kb between the Δ67P1 strain (lanes A and C) and the transformant disrupted by egl3 (FIG. 18, lanes B and D)
(The size of the inserted pyr4 fragment). Transformants containing the disrupted egl3 gene shown in FIG. 18 (lanes B and D)
And F) were designated A22. The transformant identified in FIG. 18 cannot produce CBHI, CBHII or EGII.

Example 28 Construction of pPΔEGI-1 As described in Example 12, e of T. reesei strain RL-P37
The gl1 gene was obtained as a Hind III fragment of genomic DNA. This fragment was inserted into the HindIII site of pUC100 (a derivative of pUC18, Yanishuperon et al., 1985, gene 33: Bal II, C
103-1 with oligonucleotide inserted into multiple cloning site adding laI and XhoI restriction sites
19). Using methodologies known in the prior art,
From the position near the center of the EGI coding sequence to the 3 '
About 1 kb EcoR V fragment extending beyond the end is removed, and 3.5 kb Sca of T. reesei DNA containing the pyr4 gene is removed.
Replaced by the I fragment. The prepared plasmid was transferred to pPΔEGI-
1 (see FIG. 19).

Plasmid pPΔEGI-1 was cut with HindIII, a fragment of the egl1 gene was placed at each end, and EGI1
T.reesei with a pyr4 gene replacing part of the coding sequence
A DNA fragment consisting only of genomic DNA can be released.

Digested with Hind III of appropriate T.reesei pyr4 deficient strain
Transformation by pPΔEGI-1 leads to the incorporation of this DNA at the egll locus in a percentage of transformants. In this way, a strain that cannot produce EGI is obtained.

Example 29 Construction of pΔEGIpyr-3 and Conversion of a Py4 Deficient Strain of T. reesei The expectation that the EGI gene can be inactivated using the method outlined in Example 28 is enhanced by this experiment. In this case, the plasmid, pΔEGIpyr-, is similar to pPΔEGI-1, except that the Aspergillus niger pyr4 gene replaces the T. reesei pyr4 gene as a selectable marker.
3 was constructed. In this case, the egl1 gene is Hind of pUC100
It was present again as a 4.2 kb Hind III fragment inserted at the III site. During the construction of pPΔEGI-1 (see Example 28)
The same internal 1 kb EcoR V fragment was removed,
The fragment was replaced by a 2.2 kb fragment containing the cloned A. niger pyrG gene (Wilson et al., 198
8, Nucl.Acids Res.16p.2339). Transformation of a pyr. deficient strain of T. reesei with pΔEGIpyr-3 (strain GC69) released the fragment containing the pyrG gene having the flank region from either end of the egl1 locus by cleavage with HindIII. Later, the egl1 gene was led to a disrupted transformant. These transformants were cut with HindIII and probed with radiolabeled pΔEGIpyr-3.
Was identified by Southern analysis. In an unconverted strain of T. reesei, the egl1 gene is present on a 4.2 kb Hind III fragment of DNA, and the pattern of this hybridization is shown in FIG. However, following the deletion of the egll gene by incorporation of the desired fragment from pΔEGIpyr-3, this 4.2 kb fragment disappeared, and the larger size of about 1.2
kb, FIG. 20, lane A. Figure 20
As shown in lane B, lane B is an example of a transformant in which integration of a single copy of pΔEGIpyr-3 occurred at a site in the genome other than the egl1 locus.

Example 30 Transformation of T. reesei with pPΔEGI-1 of a strain that cannot produce CBHI, CBHII, EGI and EGII According to the method outlined in Example 1, strain A22 (Example 27)
) Is obtained. This strain was previously known as Hind
III and converted by pPΔEGI-1 which releases a DNA fragment consisting only of T.reesei genomic DNA having a fragment of the egl1 gene at either end with a portion of the EGI coding sequence replaced by the pyr4 gene .

Stable pyr4 + transformants are selected and total DNA is isolated from the transformants. DNA is probed with ³² P-labeled pPΔEGI-1 after Southern analysis to identify transformants. Where contains the pyr4 gene and the egl1 sequence
A fragment of DNA is integrated at the egl1 locus, resulting in fragmentation of the EGI coding sequence. The transformants identified were CBHI, CBHI
Inability to produce I, EGI and EGII.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification number Agency reference number FI Technical indication (C12N 15/09 ZNA C12R 1: 885) (72) Inventor Weiss, Jeffrey El United States of America California 94117 San Francisco Buena Vista 457 (56) References Table 6-502223 (JP, A) Table 6-502226 (JP, A)

Claims (7)

(57) [Claims] 1. An improved method of treating a cotton-containing fabric with a fungal cellulase composition, wherein said improvement comprises one or more of:
Using a fungal cellulase composition comprising an EG type component and one or more CBH type I components, wherein the cellulase composition is a protein of all EG type components for all CBH type I components greater than 5: 1 A method comprising having a weight ratio. 2. An improved method of treating a cotton-containing fabric with an aqueous fungal cellulase solution, said method being carried out by stirring under conditions to achieve a cascade effect of said cellulase solution over said fabric. But one or more
Using a fungal cellulase composition comprising an EG type component and one or more CBH type I components, wherein the cellulase composition is a protein of all EG type components for all CBH type I components greater than 5: 1 A method comprising having a weight ratio. 3. The method of claim 2, wherein said fungal cellulase composition comprises one or more
The cellulase composition comprising an EG-type component and one or more CBH-type components, wherein the cellulase composition has a protein weight ratio of all EG-type components to all CBH-type components of greater than 5: 1. 3. The method according to claim 1 or 2. 4. The method of claim 3, wherein said fungal cellulase composition has a protein weight ratio of all EG-type components to all CBH-type components of greater than 10: 1.
The method described in the section. 5. The method of claim 1 wherein said fungal cellulase composition comprises at least about 20 weight percent of an EG-type component, based on the total weight of proteins in said cellulase composition. The method described in the section. 6. A cotton-containing fabric prepared by the method according to claim 1. 7. A cotton-containing fabric prepared by the method according to claim 2.