# Patent

## Title
Cellulase preparation containing nonionic surfactant and method of treating fiber

## Abstract

The present invention relates to a cellulase preparation comprising nonionic surfactants together with endoglucanases derived from Zygomycetes; a detergent composition comprising the above cellulase preparation with detergent components; a method of treating fabric which comprises treating cellulose-containing fabric with the above cellulase preparation so as to improve the properties of the fabric; a method of deinking waster paper which comprises a step of treating waste paper with the above cellulase preparation together with a deinking agent; and a method of improving the freeness of paper pulp which comprises a step of treating paper pulp with the above cellulase preparation.

## Background
TECHNICAL FIELD
The present invention relates to a cellulase preparation comprising a nonionic surfactant(s) by which endoglucanase activity has improved; a detergent comprising the cellulase preparation; and a method of treating fabric, which uses the cellulase preparation.
BACKGROUND ART
Cellulase has three types of enzyme activities: cellobiohydrolase activity which hydrolyzes the solid crystal regions of cellulose from the nonreduced end in an exo manner so as to generate cellobiose; endoglucanase activity which hydrolyzes the amorphous regions of cellulose in an endo manner so as to make cellulose molecules into low molecular weight molecules and to generate various types of cellooligosaccharides; and β-glucosidase activity which decomposes cellobiose or cellooligosaccharide into glucose. Of the enzymes, when endoglucanase exerts high activity, cellulase is advantageously used to treat fabric.
To impart certain desired properties to cellulose-containing fabric, the fabric has conventionally been treated with cellulase. For example, in the textile industry, treatment with cellulase is carried out to improve the touch and appearance of cellulose-containing fabric, or to impart “stonewash” appearance to colored cellulose-containing fabric thereby providing the fabric with localized color variation (EP Patent No. 307,564).
It is known that fuzz is developed on colored cellulose-containing fabric by repeated washing and that it blurs the color of the colored fabric. A cellulase-containing detergent removes such fuzz and makes the color of the fabric clear (color clarification) (EP Patent No. 220,016), and therefore detergents containing cellulase are currently on the market mainly in Europe and the United States.
In such textile processing, cellulase derived from wood-rotting fungi such as Trichoderma and Humicola is mainly used. Such cellulase is used as a mixture comprising multiple cellulase components which is obtained by processing a culture filtrate of microorganisms having cellulolytic activity. However, in order to achieve greater economy, a cellulase preparation obtained by isolating from cellulase components, only endoglucanase which largely acts on fabric treatment, and genetically enhancing it, has recently been used. Examples of such endoglucanase with high activity include: EGV (JP Patent Publication (PCT Translation) No. 5-509223) and NCE4 (WO98/03640) derived from Humicola insolens, which strongly act on cotton fabrics; RCE I, RCE II and RCE III derived from Rhizopus oryzae, which strongly act on lyocell fabrics; MCE I and MCE II derived from Mucor circinelloides; and PCE I derived from Phycomyces nitens (WO00/24879).
In order to improve the effects of cellulase, the combined use of additives has also been attempted. For example, JP Patent Publication (PCT Translation) No. 5-507615 describes that a water-soluble polymer such as polyvinylpyrrolidone, polyvinyl alcohol and polyacrylamide enhances the effects of Humicola insolens-derived cellulase and improves its activity of removing fuzz from colored fabric. Moreover, it is known that CMCase activity in the culture solution of Trichoderma viride is improved by the addition of Tween 20 (Ooshima, H. et al., Biotechnology and Bioengineering 28: 1727–1734, 1986).
However, the cellulases used for the above-described purposes are all expensive. Therefore, in order to achieve an industrial level application, further improvement of endoglucanase activity is desired, so that the above effects of cellulase can be more efficiently exerted.
DISCLOSURE OF THE INVENTION
It is therefore the object of the present invention to provide a cellulase preparation having improved endoglucanase activity, which can be used for the purpose that the fabric treatment for improving cellulose-containing fabric such as the removal of fuzz can be carried out efficiently and economically.
As a result of intensive studies directed towards the above object, the present inventors have found that a nonionic surfactant enhances the effects of Zygomycetes-derived endoglucanases such as RCE I, MCE I and PCE I, at rates far higher than Trichoderma- or Humicola-derived endoglucanase, thereby completing the present invention.
That is to say, the present invention relates to the following (1) to (5):

    (1) A cellulase preparation comprising nonionic surfactant(s) together with endoglucanase(s) derived from Zygomycetes,
    (2) A detergent composition obtained by blending said cellulase preparation with detergent components,
    (3) A method of treating fabric which comprises treating cellulose-containing fabric with said cellulase preparation so as to improve the properties of the fabric,
    (4) A method of deinking waste paper comprising a step of treating waste paper with said cellulase preparation together with a deinking agent, and
    (5) A method of improving the freeness of paper pulp comprising a step of treating paper pulp with said cellulase preparation.

[1] Cellulase Preparation



The cellulase preparation of the present invention comprises endoglucanase(s) derived from Zygomycetes, and nonionic surfactant(s).
In the present invention, the term, endoglucanase, is used to mean endo-1,4-β-glucanase EC3.2.1.4, and it has the activity to hydrolyze β-1,4-glucopyranosil bonds of β-1,4-glucan.
Examples of endoglucanases derived from Zygomycetes used in the present invention include endoglucanases derived from Rhizopus sp., Phycomyces sp. or Mucor sp. Specific examples of such endoglucanases used herein include RCE I, RCE II, RCE III, MCE I, MCE II and PCE I proteins having amino acid sequences as shown in SEQ ID NOS: 1 to 6, respectively, which are disclosed in WO00/24879.
RCE I, II and III are derived from the Rhizopus oryzae CP96001 strain, which has been deposited under the terms of the Budapest Treaty with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), [the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, the former address: Higashi 1-1-3, Tsukuba, Ibaraki, Japan] under accession No. FERM BP-6889 on Apr. 21, 1997. MCE I and II are derived from the Mucor circinelloides CP99001 strain, which has been deposited with the same international depositary authority, that is, the National Institute of Advanced Industrial Science and Technology, an Independent. Administrative Institution under the Ministry of Economy, Trade and Industry, at the AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), [the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, the former address: Higashi 1-1-3, Tsukuba, Ibaraki, Japan] under accession No. FERM BP-6890 on Jul. 2, 1999. Moreover, PCE I was derived from the Phycomyces nitens CP99002 strain, which has been deposited with the same international depositary authority under accession No. FERM BP-6891 on Jul. 2, 1999.
In the present invention, the above-stated RCE I, RCE II, RCE III MCE I, MCE II and PCE I proteins include modified proteins and homologues thereof. The term “modified protein” is used herein to mean a protein having an amino acid sequence comprising an addition, insertion, deletion or substitution of one or more amino acids (for example, one to several tens of, specifically, one to approximately fifty, preferably, one to approximately thirty, and more preferably, one to approximately nine amino acids) with respect to the amino acid sequence of each of the above mentioned RCE I, RCE II, RCE III, MCE I, MCE II and PCE I, and having endoglucanase activity. The term “homologue” is used herein to mean a protein having an amino acid sequence encoded by a gene (nucleotide sequence) complementary to a gene (nucleotide sequence) “hybridizing under stringent conditions” with a gene (nucleotide sequence) encoding the amino acid sequence of each of the above mentioned RCE I, RCE II, RCE III, MCE I, MCE II and PCE I, and having endoglucanase activity. Herein, the term “under stringent conditions” is used to mean conditions in which a probe comprising a nucleotide sequence encoding a part or all of the amino acid sequences of RCE I, RCE II, RCE III, MCE I, MCE II or PCE I, or the amino acid sequences of modified proteins thereof would hybridize with a gene encoding a homologue, but the same probe does not hybridize with an endoglucanase NCE 4 gene (SEQ ID NO: 7) as disclosed in WO98/03640 or endoglucanase SCE 3 gene (SEQ ID NO: 8) as disclosed in WO98/54322 (it should be noted that the amount of DNA used herein is equivalent to the amount of each of the NCE 4 gene, the SCE 3 gene and a gene encoding the homologue.) More specifically, it means conditions in which, using as a probe a full-length DNA sequence encoding the amino acid sequence of labeled RCE I, pre-hybridization is carried out at 42° C. for 1 hour according to the method of the ECL direct DNA/RNA labeling and detection system (Amersham), then the above probe is added thereto followed by hybridization at 42° C. for 15 hours, and thereafter, the resultant product is washed twice with a solution containing 0.4% SDS, 6 M urea and 0.5×SSC (SSC; 15 mM trisodium citrate, 150 mM sodium chloride) at 42° C. for 20 minutes, and finally followed by washing the product twice with 5×SSC at room temperature for 10 minutes.
An example of such a modified protein or homologue includes a protein comprising an amino acid sequence having preferably 80% or more homology, more preferably 90% or more homology, further more preferably 95% or more homology, and most preferably 98% or more homology with the amino acid sequence of RCE I, RCE II, RCE III, MCE I, MCE II or PCE I. The above-stated values of homology may be calculated by a homology search program which is well known to a person skilled in the art, but the values are preferably calculated using a default (initialized) parameter of FASTA3 [Science, 227, 1435–1441 (1985); Proc. Natl. Acad. Sci. USA, 85, 2444–2448 (998)
The nonionic surfactant included in the cellulase preparation of the present invention means a surfactant whose hydrophilic group is a nonionic group. Examples of such a nonionic surfactant include polyoxyethylene alkylether, polyoxyethylene alkylphenylether, polyoxyethylene monofatty acid ester, polyoxyethylene sorbitan monofatty acid ester, sorbitan monofatty acid ester, polyethylene glycol, glycerol monofatty acid ester, polyglycerine fatty acid ester, alkyl glycoside, esters of polyethoxylated alkyl glycosides, alkyl dimethyl amine oxide, fatty acid diethanol amide, polyoxyethylene alkylamine, terephthalic acid tetraethylene glycol polymer, alkyl polyethylene glycol ether, nonyl phenol polyethylene glycol ether, and fatty acid ester of sucrose or glucose. These nonionic surfactants can be used alone or in combination with other nonionic surfactants.
Moreover, the cellulase preparation of the present invention may comprise components which are conventionally contained in cellulase preparations such as excipients and preservatives. The form of the cellulase preparation may be solid or liquid, and specific examples of the form include powder, particulate, granule, non-dusting granule and liquid formulation.
A non-dusting granule that is one form of cellulase preparation can be produced according to the common dry granulation method. That is to say, powder cellulase enzyme is mixed with one or several types selected from a group comprising inorganic salts such as sodium sulfate and sodium chloride which are neutral and do not have an effect on endoglucanase activity; minerals such as bentonite and montmorillonite which do not have an effect on endoglucanase activity; and neutral organic matters such as starch and powder cellulose. Thereafter, the powders or the finely suspended suspension of one or several types of the above-described nonionic surfactants which improve the effects of endoglucanase are added to the mixture, and the thus obtained product is then fully mixed or kneaded. Depending on the situation, a synthetic polymer such as polyethylene glycol and natural polymer such as starch, which binds solids, is optionally added to the mixture and further kneaded. Thereafter, granulation is carried out by extrusion molding, using, for example, a disk pelleter, and the obtained molded material is then converted into a spherical form using a marumerizer followed by drying, so that non-dusting granules can be produced. Naturally, it is also possible to coat the surface of granules with a polymer or the like to control the permeation of oxygen or water. This time, one or multiple nonionic surfactants which improve the effect of endoglucanase are added to the above cellulase preparation at a ratio of 0.1 to 50% by weight, preferably 0.1 to 30% by weight, and more preferably 1 to 20% by weight.
On the other hand, the liquid preparation can be prepared by blending an endoglucanase stabilizer such as a synthetic and natural polymer with a cellulase solution, and adding inorganic salts or a synthetic preservative, as necessary. This time, one or multiple nonionic surfactants which improve the effect of endoglucanase can also be added. As in the case of the non-dusting granule, one or multiple nonionic surfactants which improve the effect of endoglucanase are added to the above cellulase preparation at a ratio of 0.01 to 50% by weight, preferably 0.1 to 30% by weight, and more preferably 1 to 20% by weight.
[2] Detergent Composition
The above described cellulase preparation of the present invention is blended with known detergent components such as builders, bleaching agents, bleaching activators, corrosion inhibitors, sequestering agents, stain dissociating polymers, aromatics, other enzymes, enzyme stabilizers, formulation assistants, fluorescent brightening agents and foaming promoters so that a detergent composition can be produced.
The present detergent composition relates to the granular soil removal, the color clarification, the defuzzing, the depilling and the reduction of stiffness, and these can be improved by the composition of the present invention.
[3] Method of Treating Fabric
The method of treating fabric of the present invention comprises treating cellulose-containing fabric with the above cellulase preparation.
The following properties of cellulose-containing fabric can be improved by the present fabric treatment method:

    (1) Removal of fuzz (reduction of the rate of the formation of fuzz, and reduction of fuzz),
    (2) Color clarification of colored cellulose-containing fabric,
    (3) Providing of localized color variation to colored cellulose-containing fabric, that is, providing of stonewash-like appearance and texture to colored cellulose-containing fabric, typically jeans,
    (4) Enhancement of the touch and appearance of fabric by reducing weight, and
    (5) Softening of fabric (reduction of stiffness).


The above method of treating fabric can be carried out typically during washing, but it can also be carried out during soaking or rinsing. Specifically, the method of treating fabric of the present invention can be carried out by adding the cellulase preparation of the present invention into water in which fabric is or will be soaked.
Conditions such as contact temperature and the amount of endoglucanase may appropriately be determined, taking into consideration various other conditions. For example, in the case of reducing the rate of the formation of fuzz or reducing fuzz of the cellulose-containing fabric, the fabric can be treated at a temperature of approximately 30° C. to 60° C., using 10 to 10,000 mg/L of nonionic surfactants and endoglucanases in a protein concentration of 0.05 to 20 mg/L.
In the case of providing the colored cellulose-containing fabric with localized color variations, the fabric can be treated at a temperature of approximately 30° C. to 60° C., using 10 to 10,000 mg/L of nonionic surfactants and endoglucanases in a protein concentration of 0.1 to 30 mg/L.
In a processing of reducing weight which is directed towards the improvement of the touch and appearance of the cellulose-containing fabric, the fabric can be treated at a temperature of approximately 30° C. to 60° C., using 10 to 10,000 mg/L of nonionic surfactants and endoglucanases in a protein concentration of 0.2 to 50 mg/L.
In any of the above cases, the nonionic surfactant may be dissolved or suspended in water.
The protein concentration of each type of endoglucanase is measured by HPLC analysis using TSKgel TMS-250 column (4.6 mm I.D.×75 cm) (TOSOH Corporation). The HPLC analysis involves loading acetonitrile in 0.05% TFA (trifluoroacetic acid) with a linear concentration gradient of 0% to 80% at a flow rate of 1.0 ml/min so as to elute each type of endoglucanase, and calculating the protein concentration from the peak area at UV 280 nm. A purified NCE4, the protein concentration of which is previously determined by a Protein Assay Kit (BioRad Laboratories), is subjected to the HPLC analysis in the same manner as above, so that it is used as a standard. According to the method described in International Publication No. WO98/03640, the purified NCE4 is obtained by culturing Humicola insolens [Humicola insolens MN200-1 which was deposited under accession No. FERM BP-5977 (original accession No. FERM-15736, original accession date: Jul. 15, 1996) with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology], and purifying the obtained culture. As a standard for the determination of a protein concentration in the Protein Assay Kit, Albumin Standard (Bovin serum albumin, fraction V, PIERCE) is used.
[4] Method of Deinking Waste paper
The method of deinking waste paper of the present invention comprises treating waste paper with the above-described cellulase preparation together with a deinking agent.
Specifically, the present method can be carried out by treating waste paper with the cellulase preparation of the present invention together with a deinking agent in a deinking step in a process of producing recycled paper from waste paper. The present method enables the deinking of waste paper, thereby improving the whiteness of waste paper. Waste paper which is the target of the present method include all types of common waste paper such as used news paper, used magazine paper and low to middle grade of printed used paper comprising mechanical pulp and/or chemical pulp; used wood-free paper comprising chemical pulp; and printed waste paper thereof such as coating paper. The above-described deinking agent means an agent used in the deinking of waste paper, and examples of such a deinking agent include alkali such as sodium chloride and sodium carbonate, sodium silicate, hydrogen peroxide, phosphate, anionic or nonionic surfactant, scavenger such as oleic acid, assistant agents such as pH stabilizer, chelating agent and dispersing agent, and others.
[5] Method of Improving Freeness of Paper Pulp
The method of improving the freeness of paper pulp of the present invention comprises treating paper pulp with the above-described cellulase preparation.
Specifically, the present method can be carried out by treating paper pulp with the cellulase preparation of the present invention. Examples of paper pulp which can be the target of the present method include waste paper pulp, recycled paperboard pulp, kraft pulp, sulfite pulp, thermo-mechanical treatment pulp, and other high-yield pulp.
This specification includes the contents as disclosed in the specification of Japanese Patent Application No. 2000-343921, which is a priority document of the present application.


BEST MODE FOR CARRYING OUT THE PRESENT INVENTION
The present invention is further described in the following examples. However, the examples are provided for illustrative purposes only, and are not intended to limit the scope of the invention.
Hereinafter, the term “endoglucanase activity” means CMCase activity. Moreover, regarding the “CMCase activity,” when a solution comprising cellulase enzyme and carboxymethylcellulose (CMC, Tokyo Kasei Kogyo Co., Ltd.) is incubated for a certain time period and the amount of reducing sugar released is measured, the amount of enzyme producing the reducing sugar corresponding to 1 μlmol of glucose per minute, is defined as 1 unit.
EXAMPLE 1
Comparison Among Improvement Ratios of Fuzz-Removing Activities of Various Types of Cellulases by Addition of Nonionic Surfactant
The cultivation of Rhizopus oryzae, Mucor circinelloides and Phycomyces nitens, and the purification of RCE I, MCE I and PCE I endoglucanases from the cultures, were carried out by the method described in International Publication No. WO00/24879.
The cultivation of Humicola insolens [Humicola insolens MN200-1 which was deposited under accession No. FERM BP-5977 (original accession No. FERM P-15736, original accession date: Jul. 15, 1996) with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology], and the purification of NCE4 endoglucanase from the culture, were carried out by the method described in International Publication No. WO98/03640.
The cultivation of Trichoderma viride [Trichoderma viride MC300-1 which was deposited under accession No. FERM BP-6047 (original accession No. FERM P-15842, original accession date: Sep. 9, 1996) with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology] was carried out by the method described in International Publication No. WO98/54332.
Fuzz-removing treatment from a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. was dyed in brown by reactive dyeing in Tsuyatomo-Senko), was carried out using the obtained culture supernatant and uniformly purified various endoglucanases under the following conditions.
(Test Conditions)


    Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG., Japan)
    Temperature: 40° C.
    Time: 120 minutes
    Amount of reaction solution: 40 ml
    Reaction pH: only the Trichoderma viride culture supernatant was adjusted at pH 4 (20 mM acetate buffer), all other enzyme solutions were adjusted at pH 6 (5 mM phosphate buffer) for reaction. All the buffers were prepared using deionized water.
    Type and amount of nonionic surfactant: 100 μg/ml polyoxyethylene lauryl ether (NOF Corporation, product name: NissanNonion K-220, number of oxyethylene units added: 20, HLB: 16.2)


To the treating solution were added four of about 16 g rubber balls together with the enzyme solution.
The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of both cases of adding and not adding the nonionic surfactant. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz in the case of not adding the nonionic surfactant by the amount in the case of adding the nonionic surfactant, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the nonionic surfactant. The results are shown in Table 1.









TABLE 1







Improvement ratio of



fuzz-removing activity by addition



of nonionic surfactant (fold)



















Humicola insolens culture
1.2



supernatant



Trichoderma viride culture
1.33



supernatant



Purified NCE 4
1.5



Purified RCE I
4.0



Purified MCE I
6.0



Purified PCE I
4.0










From the results of Table 1, it is found that the fuzz-removing activity of RCE I, MCE I and PCE I, which are endoglucanases derived from Zygomycetes, is improved by the addition of the nonionic surfactant at a level far higher than other enzymes including cellulase derived from Humicola insolens or Trichoderma viride, which are previous findings.
EXAMPLE 2
Improvement Effect of Fuzz-Removing Activity of RCE I Expressed in Humicola by Addition of Various Nonionic Surfactants
RCE I endoglucanase was expressed in Humicola insolens according to the method described in Examples D3 and 4 of International Publication No. WO00/24879. Fuzz-removing treatment from a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. was dyed in brown by reactive dyeing in Tsuyatomo-Senko), was carried out using the obtained culture supernatant under the following conditions.
(Test Conditions)


    Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG., Japan)
    Temperature: 40° C.
    Time: 120 minutes
    Amount of reaction solution: 40 ml
    Reaction pH: pH 6 (5 mM phosphate buffer prepared with deionized water)
    Amount of nonionic surfactants: 100 μg/ml
    Type of nonionic surfactants: Refer to Table 2 as described below.


To the treating solution were added four of about 16 g rubber balls together with the enzyme solution.









TABLE 2





Type of surfactants
Product names
Manufacturing companies







Polyoxyethylene lauryl
Brij-35
Wako Pure Chemical


ether (number of oxyethy-

Industries, Ltd.


lene units added: 4)


Polyoxyethylene lauryl
K-220
NOF Corporation


ether (number of oxyethy-


lene units added: 20)


Polyoxyethylene cetyl ether
P-208
NOF Corporation


(number of oxyethylene


units added: 8)


Polyoxyethylene nonyl
Nonipol 100
Sanyo Chemical


phenyl ether

Industries, Ltd.


Polyoxyethylene octyl
Triton X100
Wako Pure Chemical


phenyl ether

Industries, Ltd.


Polyoxyethylene sorbitan
Tween 80
Wako Pure Chemical


monooleate

Industries, Ltd.


Polyethylene glycol 4000
PEG 4000
Wako Pure Chemical




Industries, Ltd.


Terephthalic acid tetra-
FR 550
Goo Chemical Co., Ltd.


ethylene glycol polymer









The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of both cases of adding and not adding each nonionic surfactant. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz in the case of not adding the nonionic surfactant by the amount in the case of adding the nonionic surfactant, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the nonionic surfactant. The results are shown in Table 3.








TABLE 3






Improvement ratio of fuzz-removing



activity by addition of


Type of nonionic surfactants
nonionic surfactants (fold)
















Polyoxyethylene lauryl ether
3.0


(number of oxyethylene units


added: 4)


Polyoxyethylene lauryl ether
3.0


(number of oxyethylene units


added: 20)


Polyoxyethylene cetyl ether
2.0


(number of oxyethylene units


added: 8)


Polyoxyethylene nonyl
3.0


phenyl ether


Polyoxyethylene octyl
2.0


phenyl ether


Polyoxyethylene sorbitan
2.0


monooleate


Polyethylene glycol 4000
2.0


Terephthalic acid tetraethylene
2.5


glycol polymer









From the results of Table 3, it is found that the fuzz-removing activity of the culture supernatant obtained by expressing and secreting RCE I in Humicola insolens was improved by any of the above nonionic surfactants.
EXAMPLE 3
Improvement Effect of Fuzz-Removing Activity of RCE I Expressed in Humicola by Addition of Nonionic Surfactants with Various Concentrations
RCE I endoglucanase was expressed in Humicola insolens according to the method described in Examples D3 and 4 of International Publication No. WO00/24879. Fuzz-removing treatment from a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. was dyed in brown by reactive dyeing in Tsuyatomo-Senko), was carried out using the obtained culture supernatant under the following conditions.
(Test Conditions)


    Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MPG., Japan)
    Temperature: 40° C.
    Time: 120 minutes
    Amount of reaction solution: 40 ml
    Reaction pH: pH 6 (5 mM phosphate buffer prepared with deionized water)
    Amount of nonionic surfactant: 10 to 10,000 μg/ml
    Type of nonionic surfactant: polyoxyethylene lauryl ether (NOF Corporation, product name: NissanNonion K-220, number of oxyethylene units added: 20, HLB: 16.2)


To the treating solution were added four of about 16 g rubber balls together with the enzyme solution.
The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in the case of adding the nonionic surfactant in each concentration. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz in the case of not adding the nonionic surfactant, by the amount in the case of adding the nonionic surfactant in each concentration, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the nonionic surfactant in each concentration. The results are shown in Table 4.








TABLE 4






Improvement ratio of fuzz-removing


Additive amount of nonionic
activity by addition of


surfactant (μg/ml)
nonionic surfactant (fold)
















10
1.5


20
2.0


50
2.5


100
3.0


200
3.0


400
3.0


1000
3.0


2000
2.0


3000
1.5


5000
1.5


10000
1.5









From the results of Table 4, it is found that the fuzz-removing activity of the culture supernatant obtained by expressing and secreting RCE I in Humicola insolens was improved by the addition of the nonionic surfactant having a wide range of concentration from 10 to 10,000 μg/ml.
EXAMPLE 4
Production of RCE I Cellulase Preparation Comprising Nonionic Surfactant
After mixing the following raw materials, an appropriate amount of water was added thereto, and the mixture was kneaded. The obtained product was subjectetd to a disk pelleter for molding, and the product obtained by injection molding was converted in a particle form using a marumerizer (Fuji Paudal Co., Ltd.) followed by drying and sieving the product so as to obtain a granulated product.












Mixing



ratio


(Raw materials)
(%)







S-220 (nonionic surfactant manufactured by NOF Corporation)
  10%


RCE I cellulase powder product
  10%


Magnesium chloride (Wako Pure Chemical Industries, Co., Ltd.)
 0.5%


Monopotassium phosphate (same as above)
  2%


Dipotassium phosphate (same as above)
  1%


Corn starch (Shikishima Starch Co.)
76.5%









The RCE I cellulase powder product was prepared by concentrating the culture supernatant of RCE I expressed in Humicola insolens using ultrafiltration, according to the method described in Examples D3 and 4 of International Publication No. WO00/24879, followed by spray drying.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.
INDUSTRIAL APPLICABILITY
The present invention provides a cellulase preparation having dramatically improved Zygomycetes-derived endoglucanase activity by adding a nonionic surfactant into the preparation. If the present cellulase preparation is used in the treatment of fabric such as the reduction of fuzz of cellulose-containing fabric, the improvement of touch and appearance, the color clarification, localized color variation or softening, the deinking of waste paper, or the processing of improving the freeness of paper pulp, each of the above treatments can be carried out with a less amount of enzyme, thereby significantly reducing cost.

## Claims

1. A cellulase preparation for enhanced fuss-removing activity comprising nonionic surfactant(s) together with a protein comprising the amino acid sequence of SEQ ID NO: 1.

2. A cellulase preparation for enhanced fuss-removing activity comprising nonionic surfactant(s) together with protein(s) encoded by
DNA encoding the amino acid sequence of SEQ ID NO: 1 or
DNA which is fully complementary to a DNA hybridizing to a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 under conditions in which, using as a probe a full-length DNA sequence encoding the amino acid sequence of labeled RCE I, wherein pre-hybridization is carried out at 42° C. for 1 hour according to the method of the electrochemiluminescent (ECL) direct DNA/RNA labeling and detection system (Amersham), then the probe is added thereto followed by hybridization at 42° C. for 15 hours, and thereafter, the resultant product is washed twice with a solution containing 0.4% SDS, 6Murea and 0.5×SSC (SSC; 15 mM trisodium citrate, 150 mM sodium chloride) at 42° C. for 20 minutes and finally followed by washing the product twice with 5×SSC at room temperature for 10 minutes and which encodes a protein having endoglucanase activity.

3. The cellulase preparation according to claim 1 wherein the nonionic surfactant is polyoxyethylene alkylether, polyoxyethylene alkylphenylether, polyoxyethylene monofatty acid ester, polyoxyethylene sorbitan monofatty acid ester, sorbitan monofatty acid ester, polyethylene glycol, glycerol monofatty acid ester, polyglycerine fatty acid ester, alkyl glycoside, esters of polyethoxylated alkyl glycosides, alkyl dimethyl amine oxide, fatty acid diethanol amide, polyoxyethylene alkylamine, terephthalic acid tetraethylene glycol polymer, alkyl polyethylene glycol ether, nonyl phenol polyethylene glycol ether, or fatty acid ester of sucrose or glucose.

4. The cellulase preparation according to claim 1 comprising 0.1 to 50% by weight of nonionic surfactant(s).

5. The cellulase preparation according to claim 1, which is a non-dusting granule or stabilized liquid.

6. A detergent composition comprising the cellulase preparation according to claim 1 with detergent components.

7. A method of treating fabric which comprises treating cellulose-containing fabric with the cellulase preparation according to claim 1 so as to improve the properties of the fabric.

8. The method according to claim 7 wherein the improvement of the properties of the fabric is the color clarification.

9. The method according to claim 7 wherein the improvement of the properties of the fabric is the removal of fuzz.

10. The method according to claim 7 wherein the improvement of the properties of the fabric is the providing of stonewash-like appearance and texture.

11. The method according to claim 7 wherein the improvement of the properties of the fabric is the improvement of touch and appearance.

12. The method according to claim 7 wherein the improvement of the properties of the fabric is softening of the fabric.

13. The method according to claim 7 wherein nonionic surfactant(s) are present in a concentration of 10 to 10,000 mg/L in a reaction system.

14. The method according to claim 7 wherein the method is carried out through a step of soaking, washing or rinsing the fabric.

15. A method of deinking waste paper comprising a step of treating waste paper with the cellulase preparation according to claim 1 together with a deinking agent.

16. A method of improving the freeness of paper pulp comprising a step of treating paper pulp with the cellulase preparation according to claim 1.

17. The cellulase preparation according to claim 2 wherein the nonionic surfactant is polyoxyethylene alkylether, polyoxyethylene alkylphenylether, polyoxyethylene monofatty acid ester, polyoxyethylene sorbitan monofatty acid ester, sorbitan monofatty acid ester, polyethylene glycol, glycerol inonofatty acid ester, polyglycerine fatty acid ester, alkyl glycoside, eaters of polyethoxylatecl alkyl glycosides, ailcyl dimethyl amine oxide, fatty acid diethanol amide, polyoxyothylene alkylamine, terephthalic acid tetraethylene glycol polymer, alkyl polyethylene glycol ether, nonyl phenol polyethylene glycol ether, or fatty acid ester of sucrose or glucose.

18. The cellulase preparation according to claim 2 comprising 0.1 to 50% by weight of nonionic surfactant(s).

19. The cellulase preparation according to claim 2, which is a non-dusting granule or stabilized liquid.

20. A detergent composition comprising the cellulase preparation according to claim 2 with detergent components.

21. A method of treating fabric which comprises treating cellulose-containing fabric with the ceflulase preparation according to claim 2 so as to improve the properties of the fabric.

22. The method according to claim 21 wherein the improvement of the properties of the fabric is the color clarification.

23. The method according to claim 21 wherein the improvement of the properties of the fabric is the removal of fuzz.

24. The method according to claim 21 wherein the improvement of the properties of the fabric is the providing of stonewash-like appearance and texture.

25. The method according to claim 21 wherein the improvement of the properties of the fabric is the improvement of touch and appearance.

26. The method according to claim 21 wherein the improvement of the properties of the fabric is softening of the fabric.

27. The method according to claim 21 wherein nonionic surfactant(s) are present in a concentration of 10 to 10,000 mg/b in a reaction system.

28. The method according to claim 21 wherein the method is carried out trough a step of soaking, washing or rinsing the fabric.

29. A method of deinking waste paper comprising a step of treating waste paper with the cellulase preparation according to claim 2 together with a deinking agent.

30. A method of improving the freeness of paper pulp comprising a step of treating paper pulp with the celiulase preparation according to claim 2.