FI119434B - Enzyme fusion proteins and their use - Google Patents

Description

119434

Enzyme fusion proteins and their use

Field of the Invention

The present invention relates to enzyme technology, and more particularly to cellulase fusion proteins comprising a catalytic domain and a cellulose binding domain. Fusion proteins can be produced by recombinant techniques using appropriate polynucleotides, expression vectors and host cells, which are also part of the invention. Fusion proteins and their enzyme preparations are useful in the treatment of cellulosic material, such as textile material. In addition, fusion proteins can be used in the pulp and paper industry, in oil extraction from plants, in detergent compositions and in improving the quality of animal feed. The invention thus further relates to a process for treating cellulosic material with a fusion protein, and in particular to processes for biofuel washing or bio-finishing of fabrics or garments, especially denim. The invention still further relates to detergent compositions and animal feed containing fusion proteins.

Background of the Invention

Cellulose is a major structural component of higher plants and occurs naturally in virtually pure form only in cotton fibers. It provides plant cells with high tensile strength, helping them to resist me-,) · * 20 caustic load and osmotic pressure. Cellulose is a straight-chain polysaccharide of glucose linked by β-1,4 bonds. In nature, cellulose: * is usually associated with lignin and hemicelluloses. Several different organisms, • •:. ·. including bacteria and fungi, break down cellulosic material in nature. Biological conversion of cellulose to glucose generally requires three major groups of enzymes, · · * .I ”, 25 cellobiohydrolases (CBH), endoglucanases (EG), and beta-glucose **“ * deases (BG).

There is a wide range of industrial applications of cellulases. In the textile • ♦: ** industry, cellulases are used in finishing denim to create a fashionable stone «« · washed appearance for denim fabrics in the bi-stone washing process. Pulp; * ··. 30 glasses are also used for example to remove lint and prevent nyp- *. ···. formation of soot on the surface of cotton garments. In the detergent industry, standing · · / ·) lulases are used to brighten colors and prevent graying and flaking of clothes. Cellulases are still used in the food industry and animal feeds and have great potential in the pulp and paper industry, for example in deinking to decolorize fiber surfaces and improve dewatering of pulp.

Industrially used cellulases are often mixtures of enzymes with a variety of activities and substrate specificities. Commercial enzyme-5 preparations often comprise all three cellulase activities i.e. CBH, EG and BG. In addition, the unique properties of each cellulase make some more suitable for certain purposes than others, and therefore efforts have been made to obtain and utilize cellulases having only the desired activities. The most widely used fungal cellulases are from Trichoderma reeseis-10. However, other sources of fungi have also been proposed, for example, in US 5,457,046.

The cellulases used to treat denim are generally divided into two main groups: acidic and neutral cellulases. Acid cellulases typically operate at pH 4.0-5.5 and neutral cellulases at pH 6-8. Sel-15 lulases having both acidic and neutral group characteristics may be referred to as hybrid cellulases. The acidic cellulases used in the biolith wash are mainly derived from Trichoderma Reese / (the sexual form Hypocrea jeco-rina) and the neutral cellulases come from a variety of fungi including Melanocarpus, Humicola, Myceliophthora, Fusarium, Acremonium and Chryso-20 spor. . T. reesei enzymes include, for example, cellulases from glycosyl hydrolase family 5 (endoglucanase II, EGII), family 7 (cello ··· j biohydrolase I, CBHI) and family 12 (endoglucanase III, EGIII; Ward et al.

: 1993) and neutral cellulases, most often endoglucanases, from family 45: ** and family 7 (Henrissat, 1991; Henrissat and Bairoch, 1993, 1996).

25 US 5 874 293 discloses an improved cellulase composition comprising increased amounts of T. reesei EGII endoglucanase for treating cellulose-containing textiles. The composition improved color properties, increased brightness and visual appearance and WO97 / 14804 discloses one 20 kDa and one 50 kDa cellulase with • φ · 30 endoglucanase activities derived from Melanocarpus a and which are particularly useful in textiles. Fusion proteins containing 20K and 50K proteins linked to a cellulose binding domain, preferably from Trichoderma Reese, are proposed for the creation of novel enzyme biomolecules. No specific examples are given or desirable characteristics. • · · * ". * 35 mouths to describe.

• * • · ·· «3 119434

However, there is still a need for improved cellulases, including endoglucanases, which are more effective in fabric treatment and other fields where cellulases are traditionally used. In particular, there is a continuing need for more efficient cellulases to improve the economics of the processes. The present invention aims to meet these needs.

In the textile industry, the '' stone-washed 'appearance or worn-out appearance has been of interest to denim producers in recent years. Conventional pumice stone washing reduces the fabric strength and loads the washing machines. The trend is towards enzymatic denim finishing processes and cellulases have been replaced or used in combination with pumice stones to give the fabric the desired "worn" look. Controlled enzyme treatments result in less damage to clothing and machines and eliminates the need for stone destruction.

A common problem with enzymatic stonewashing is re-staining caused by re-deposition of the removed indi-15 go during or after abrasion. Indigo dye redeposition reduces the desired contrast between white and indigo dyed yarns and is most easily noticed on the inside of the denim and in the inner pockets (increased blueness). On the right, this can be seen as a reduced contrast between the stained areas and the areas that have been decolourized during the biofilm wash. Back staining can be reduced by using anti-staining agents, such as nonionic ethoxylated alcohols, during processing, or by adding bleaching agents during the rinsing steps. The nature of the enzyme ·:, * · {la has an effect on back-staining. Generally, neutral cellulases reverse: less staining than acidic cellulases.

: WO97 / 09410 discloses that the addition of one type of cellulase to another stands for • «. ***. lulase with abrasive activity reduces back staining. The additional cellulase belongs to family 5 or 7 but does not itself have a significant abrasive effect. Preferably, said additional cellulase is derived from Bacillus or * ... * 30 Clostridium.

US 5,916,799 discloses cellulase compositions containing both: T: cellobiohydrolases and endoglucanases that have been subjected to limited proteolysis to separate the core and binding domains of the enzymes. Received ent-. \ # Chemical formulations were found to reduce back staining. WO94 / 07983 35 relates to the observation that redeposition of dye on a fabric during a stone washing process can be reduced by using a fungal cellulase cellulose. 119434 4 nuclei which are substantially free of cellobiohydrolase type components. WO96 / 23928 discloses the treatment of cellulose-containing fabrics with truncated cellulase enzymes. Truncated enzymes lacking the cellulose binding domain (CBD) were found to reduce discoloration and li-5 to control abrasion.

A general conclusion from the above three references could be that the cellulose binding domain has a negative effect on back dyeing. However, the present invention now provides a cellulase structure that has a low back dye property despite the presence of a cellulose binding domain 10.

Summary of the Invention

The present invention is based on the surprising finding that the action of endoglucanases could be significantly enhanced by combining with them a specific cellulose binding domain without degrading the back dyeing properties.

One object of the present invention is to provide novel endoglucanase fusion proteins with improved hydrolytic properties for use in the textile industry, particularly denim stonewashing. The fusion proteins of the invention have the advantage of being active at both acidic and neutral pH and having greatly improved potency in biofuel washing applications. When used in the treatment of denim, the fusion proteins exert their Mata - {**) staining effect. Due to the improved efficiency of the endoglucanases of the invention, the use of enzymes is significantly more economical. Additional benefits are also achieved with respect to the logistics and storage of enzyme products. . ·. 25 because smaller amounts of enzyme product are needed. Fusion proteins are. · **. also useful in detergent compositions and other applications such as the feed ♦ * industry, oil extraction from plants or the pulp and paper industry.

It is a further object of the present invention to provide polynucleotides encoding novel endoglucanase fusion proteins.

It is a further object of the present invention to provide a process for the production of fusion proteins.

. * ··. It is a still further object of the present invention to provide new • · • * ·. ·. expression vectors containing such dust nucleotides and useful for the production of endoglucanase fusion proteins, and novel hosts transformed with said 35 expression vectors.

5,119,434

It is a further object of the present invention to provide enzyme preparations containing one or more endoglucanase fusion proteins.

It is a further object of the present invention to provide methods for treating cellulosic material with a fusion protein, e.g., for use in the textile, detergent or animal feed industry, oil extraction from plants or pulp and paper industry, and particularly textile finishing, especially denim bio-stonewashing.

It is a further object of the present invention to provide animal feed or detergent compositions containing fusion proteins.

The present invention relates to a cellulase fusion protein comprising a first endoglucanase core amino acid sequence having at least 75% identity to SEQ ID NO: 2 or a fragment thereof having cellulase activity and a second amino acid sequence comprising a linker and a cellulose binding domain (CBD). at least 75% identity to SEQ ID NO: 15 or a fragment thereof having cellulose binding activity.

The invention is further directed to an isolated polynucleotide selected from the group consisting of: (a) a nucleotide sequence according to SEQ ID NO: 3 or a patent. the sequence encoding the cellulase fusion protein of claim 1, (b) the complementary strand of (a): (c) a sequence degenerate with respect to the genetic code of any of the sequences defined in (a) or (b).

jt: *; The invention still further relates to an expression vector comprising: said nucleotide sequence, and a host cell comprising an expression vector: *** and an enzyme preparation comprising a fusion protein.

· · ·

The invention also encompasses a method of producing a fusion protein, comprising the steps of transforming a host cell with said expression vector encoding a fusion protein • · f M, and culturing said host cell under conditions which allow expression and purification of said fusion protein. .

The invention still further comprises a process for treating cellulosic material, said process comprising contacting the cellulosic material with a fusion protein.

• * • · 6 119434

The invention is also directed to a bi-stone washing process comprising the step of contacting a cellulase fusion protein or enzyme preparation with a denim fabric or garments and a bio-finishing process comprising the step of contacting a cellulase fusion protein or enzyme preparation with textiles or fibers.

Finally, the invention is directed to a detergent composition comprising fusion protein and detergent adjuvants, to animal feed comprising a fusion protein, and to an Escheria coli strain bearing accession number E. coli DSM 10 18159.

Specific embodiments of the invention are set forth in the dependent claims.

Other objects, details, and advantages of the present invention will become apparent from the following drawings, detailed description, and examples.

Brief Description of the Drawings

Figure 1 illustrates a schematic representation of an expression cassette used in transformation of T. reeselh protoplasts for the production of T. aurantiacus EG28 or EG28 + CtCBD cellulases. The ce / 5A or ce / 5A_Ct ce / 7AlinkkeriCBD-20 gene is under the control of the T. reesei coliII promoter (cbhl prom) and the transcription termination is confirmed by the addition of the cbh1 terminator (cbhl term). The fV amdS gene (amdS) is included for the selection of transformants. Expression cassette for EG28 and EG28 + CtCBD production was isolated as an 8.6 kb A / of1 fragment from pALK1930 or as an 8.9 kb A / of1 fragment from pALK1948. ·· ·. . ·. 25 in order.

. ···. Figure 2 illustrates the pH dependence of heterologously produced T. aurantiacus • · EG28 cellulase by determining the culture supernatant. . en CMC as a substrate in a 10 minute reaction at 50 ° C (A). The temperature optimum of EG28 cellulase was determined at optimal pH (6.0). The reaction containing: ··· * 30 containing CMC as a substrate was carried out for 10 minutes (B). The pH and temperature optimizers of EG28 + CtCBD fusion protein were determined to be the same as wild type; ***; EG28 cellulase.

· · ·

Figure 3 shows the bi-stone washing effect of EG28 cellulase by measuring color at various temperatures and pH 6.

J · * 35 Figure 4 shows the bi-stone washing effect of EG28 + CtCBD cellulase by measuring the color at different pHs at 50 ° C.

7, 119434

Figure 5 shows the bi-stone washing effect of EG28 + CtCBD cellulase by measuring the color at various pHs at 60 ° C.

Figure 6 shows the bi-stone washing effect of EG28 + CtCBD cellulase by measuring color at different temperatures (pH 6).

Figure 7 shows the effect of the game starch temperature on the recovery of betaglucanase activity in feeds supplemented with EG28 cellulase.

Figure 8 shows the effect of the game starch temperature on the recovery of cellulase activity in feed supplemented with EG28 + CtCBD-cellulase.

DETAILED DESCRIPTION OF THE INVENTION Cellulases comprise a catalytic domain / nucleus (CD) expressing cellulase activity. In addition to the catalytic domain, the cellulase molecule may comprise one or more cellulose binding domains ("fCBDs"), also called carbohydrate binding domains / modules (CBD / CBM), which may be located at either the N or C termini of the catalytic domain. CBDs have carbohydrate-binding activity and mediate cellulase binding to crystalline cellulose, but have little or no effect on the enzyme's hydrolytic activity against soluble substrates. These two domains typically combine with a flexible and highly glycylated linker, as a linker ". Such constructs are described, for example, in Srisodsuk et al., 1993. Some naturally occurring endoglucanases and cellobiohydrolases have a cellulose V binding domain (CBD) while others do not.

Endoglucanases (EGt) are one of three cellulase types that; generally required for the biological conversion of cellulose to glucose. Endoglu-; 25 canases cleave internal beta-1,4-glucoside bonds when cellobiohydrolase. they again disrupt the disaccharide cellobiose at the end of the cellulose polymer chain and the beta-1,4-glucosidases hydrolyze the cellobiose and other short cello-oligosaccharides to glucose. "Endoglucanase" ("EG") in the context of the present invention refers to enzymes classified in E.C. 3.2.1.4:ksi. They are 1,4-beta-D-glucan-4-glucanohydrolases and catalyze endohydrolysis of 1,4-beta-D-glucosidic linkages in glucose polymers such as cellulose.

· * ": Some endoglucanases may also hydrolyze, e.g., 1,4-bonds in beta-D- ·· * glucans which also contain 1,3-bonds. They may therefore be classified as * 9 also endo-1,3 (4) - Beta-glucanases (EC 3.2.1.6) Thus, the enzyme can catalyze reactions on a variety of substrates and can fall into several classes.

8 119434

Cellulases, including endoglucanases, can also be classified into various families of glycosyl hydrolases according to their primary sequence, supported by analysis of the three-dimensional structure of some members of the family (Henrissat 1991, Henrissat and Bairoch 1993, 1996). For example, family 45 (en-5 nen celK) contains endoglucanases (EC 3.2.1.4) and family 5 (formerly known as celA) consists mainly of endoglucanases (EC 3.2.1.4). Family 7 (before the cellulase family celC) contains both endoglucanases and cellobiohydrolases. Some glycosyl hydrolases are multifunctional enzymes containing catalytic domains belonging to different families of glycosyl hydrolases. 10 For the present purposes, the endoglucanase moiety of the fusion protein belongs to family 5.

According to a preferred embodiment of the invention, the endoglucanase portion of the fusion protein is derived from a fungus, preferably of the genus Thermoascus, and more preferably of the genus Thermoascus aurantiacus. Such an endoglucanase is described, for example, in Hong et al. 2003. WO00 / 062409 proposes the use of this enzyme in feed applications because, in addition to endoglucanase, it also has beta-glucanase activity. Most preferably, the endoglucanase is derived from T. aurantiacus strain ALK04242, deposited as CBS 116239. The endoglucanase gene of said strain is inserted into pALK1926 and deposited as DSM 17326. The protein encoded by this gene is referred to herein as Ta EG28 "or simply" EG28 ".

• · ·

The "core" of endoglucanase, as used herein, refers to the enzyme • · [catalytic domain / core (CD), which expresses at least endoglucanase ·. * ·: Activity. Such a catalytic domain may be in its naturally occurring * · 25 form (i.e., intact). or it may be modified.

According to one embodiment of the invention, the endoglucanase nucleus has at least 75, 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 2 (Ta EG28). Preferably, the core comprises at least a mature protein,? corresponding to amino acids 19-334 of SEQ ID NO: 2. Signal sequence prediction was made using SignalP V3.0 (Nielsen et al., 1997; Bendt-· "* sen et al., 2004); NN value was obtained using neural networks and HMM value was used. · With hidden Markov models.

"Cellobiohydrolase" or "CBH" as used herein refers to the former. ···. to the enzymes that cleave cellulose at the end of the glucose chain and produce • · 35 mainly cellobiose. They are also called 1,4-beta-D-glucan cellobiohydro-* 'or cellulose-1,4-beta-cellobiosidase. They hydrolyze 1,4-beta-9,119,434 D-glucoside bonds at the reducing or non-reducing ends of the polymer containing said bonds, such as cellulose, to release the cellobiose.

The linker-containing CBD is preferably derived from Chaetomium thermo-philum, and in particular the 5 gene encoded cellobiohydrolase (CBHI / Cel7A) of strain ALK04265, deposited as CBS 730.95. This linker containing CBD is referred to as "CtCBD". According to a preferred embodiment of the invention, the linker and the cellulose binding domain have a sequence having at least 80, 85, 90, 95, 98 or 99% identity to SEQ ID NO: 15 (corresponding to amino acids 335-415 of SEQ ID NO: 4). According to another preferred embodiment, the second amino acid sequence comprises amino acids 335-379 of SEQ ID NO: 4.

The term "derived from" in connection with a microorganism source means that the polypeptide may be naturally produced in said particular microorganism source, or the polynucleotide encoding the polypeptide may be isolated from said 15 microorganism sources, or the term refers to a host cell into which the polypeptide encoding it does not exclude minor modifications of the sequence, e.g., by substitution, deletion, inversion and / or insertion of one or more amino acids / codons as long as the biological activity of the encoded protein is maintained.

The core and linker + CBD in the said order may also be, a fragment or variant of said sequence, wherein said fragment or variant * * ·

Khant has cellulase activity and / or cellulose binding activity. For example: a first amino acid sequence may comprise an amino acid sequence having at least 75% identity to SEQ ID NO: 2 or 8, a fragment · · ·: * 25 or a variant and a second amino acid sequence comprising an amino acid sequence having at least 75% identity. 75% identity with SEQ ID NO: 15, fragment: ***, or variant.

• · ·

In the present context, "cellulase activity" means the catalytic v. Ability to hydrolyze cellulose or its derivatives, such as endoglu, ···. Canase or betaglucanase activity. In addition to endoglucanase and / or betaglucanase activity, some cellulases may still have h ··: '· * and / or xylanase activity.

As used in the present context, the term '' identity 'is a reference, ···. global identity between the two amino acid sequences from the first 35 amino acids encoded by the corresponding gene to the last amino acid. The full-length sequence identity is measured by 10,119,434 using the Needleman-Wunsch Global Alignment Program in EMBOSS (European Molecular Biology Open Software Suite; Rice et al. 2000), version 3.0.0, with parameters EMBLOSUM62, gap penalty 10.0, extend penalty 0.5. The algorithm is described in Needleman and Wunsch (1970). One skilled in the art is aware of the fact that the results obtained using the Needleman-Wunsch algorithm are only comparable when aligning the corresponding domains of the sequence. Therefore, a comparison of e.g. cellulase sequences containing CBD or signal sequence with sequences lacking these elements is excluded as irrelevant.

According to one embodiment of the invention, the fusion protein contains an endoglucanase nucleus encoded by a gene corresponding to a gene included in E. coli DSM 17326. Preferably, the fusion protein is encoded by a fusion gene corresponding to the gene included in E. coli DSM 18159. According to a specific embodiment of the invention, the fusion protein contains an endoglucanase nucleus comprising SEQ ID NO: 2 and a linker and CBD comprising SEQ ID NO: 15. In particular, it comprises the amino acid sequence of SEQ ID NO: 4 or a fragment thereof having cellulase activity and cellulose binding activity.

The 'fragment' is understood to be a portion of a specific amino acid sequence long enough to possess the desired biological activity.

In other words, the fragment can be, for example, only the mature portion · »· of the amino acid sequence, or even a sub-sequence of the mature portion. An "amino acid sequence" which is a "variant" of a specific * · * ·] amino acid sequence means an amino acid sequence which is not!. * ·: Not identical to a specific amino acid sequence, but rather contains • ·: · * 25 at least some amino acid changes, i.e. deletions. , inversions, insertions, etc., which do not substantially affect the biological: ***: activity, when compared to the similar ··· activity of the specific amino acid sequence for the intended use. Biological activity, i.e. herein refers to cellulase activity, cellulose binding activity or both.

• *

The fusion protein of the invention can be prepared by attaching endoglucans; * ·· the backbone nucleus to the linker and the CBD moiety in the appropriate coding DNA using commonly known recombinant DNA techniques. In short, ·· *. polynucleotides encoding the fusion partners are amplified and cloned, the nucleotides can also be synthesized. The fused polynucleotide is inserted into an expression vector, transformed into a host cell, and expressed. Preferably, the linker and CBD are linked to the C-terminus of the endoglycanase nucleus.

An "expression vector" is a cloning plasmid or vector capable of expressing DNA encoding endoglucanase fusion proteins after transformation into a desired host. When using a fungal host, the gene of interest is preferably passed on to Other sequences that are part of a cloning vehicle or expression vehicle 10 may also be integrated into said DNA during the integration process, and in addition, the fungal expression vector or portions thereof may be targeted to a predetermined locus.

Preferably, the DNA encoding the endoglucanase fusion proteins is placed under the control (i.e., functionally linked to) of certain control sequences mediated by the vector, such as promoter sequences. In transformation, these control sequences integrate into the host genome with the gene of interest.

Alternatively, the control sequences may be those at the integration site.

, The sequences controlling expression of the expression vector will vary depending on whether the vector is designed to express a particular gene in an iV prokaryotic or eukaryotic host (for example, the shuttle vector may transmit the gene for selection of bacterial hosts). Expression control sequences may include transcriptional regulatory elements such as promoters, enhancer elements and transcriptional termination sequences and / or translational regulatory elements such as translational start and termination sites.

A polynucleotide molecule, such as DNA, is said to be capable of expressing a polypeptide if it contains expression control sequences, '*: ** containing transcriptional regulatory information, and such sequences ··:' ·· operably linked to a nucleotide sequence encoding a polypeptide.

A functional linkage is a linkage in which a sequence is linked to a regulatory,. Lys., Sequence (or sequences) in such a way that expression of the sequence is subjected to control or control. Two DNA: · * sequences (such as the promoter region sequence fused to the 5 'end of the 12 119434 protein encoding the sequence) are said to be operably linked if the promoter function leads to transcription.

The vectors of the invention may further comprise other functionally linked regulatory elements, such as amplifier sequences.

In a preferred embodiment, genetically stable transformants are coprostrated to integrate the DNA encoding the fusion proteins into the host chromosome by transformation with a vector that can protect sequences promoting integration of said vector into the chromosome.

Cells with DNA encoding endoglucanase fusion proteins stably integrated in their chromosome may be selected, e.g., by introducing markers, homologous or heterologous, that allow selection of host cells containing the expression vector, e.g., resistance to biocide resistance, e.g. copper, against, or markers that complement the auxotrophic mutation in the host chromosome or the like. For example, the selectable marker may be a selectable gene directly linked to the expressed DNA gene sequences or introduced into the same cell by co-transformation. Other selection systems may also be used.

When an expression vector containing the DNA encoding the fusion protein is prepared, it is introduced into the appropriate host cell by any suitable means, including transfer- ··· • ** | mation. After transformation, the recipient cells are grown in an appropriate selective medium that selects the transformed cells t · \ * ·; to grow.

Suitable expression and production host systems include, for example, fungal Trichoderma (EP 244 234) or Aspergillus hosts such as A. Oryx '*; zae or A. niger (WO 97/08325 and WO 95/33386, U.S. Patent No. 5,843,745, U.S. Patent No. 5,770,418), a production system developed or for Fusarium such as F. oxysporum (Malardier et al., 1989), developed production system. Suitable production systems for bacteria · · · l.lm 30 include BacillukseHe, e.g., B. subti- *: ** // for it, B. lichaniformisiWe, B. amyloliquefaciensiWe, or E. coli or actinomycete · * · * ··· production system for repfomyces. Suitable production systems for yeast include Saccharomycasitte, Shizosaccharomyces \\\ e or Pichia. ···. pasfer / systems developed for it. Production systems in other microbes or mammalian • · /.".t 35 hand cells or plants are also possible.

• · * * 13 119434

Expression of the cloned / cloned gene sequence (s) results in production of the desired protein or fragment of this protein. This expression may be in a continuous manner in transformed cells or in a controlled manner.

In order to obtain the enzyme preparations of the invention, hosts having the desired properties (namely hosts capable of expressing economically feasible amounts of endoglucanase fusion proteins) are cultured under suitable conditions, and the desired enzymes are preferably secreted from the host medium and cultured medium. Preferably, the host for such production is a filamentous plant such as Trichoderma or Aspergillus, and in particular T. reesei.

As used herein, "enzyme preparation" refers to any enzyme product containing at least one endoglucanase fusion protein of the invention. Thus, such enzyme preparation may be a spent culture medium or a filtrate. If desired, such preparations may be freeze-dried or the enzyme activity may otherwise be concentrated and / or stabilized for storage If required, the desired enzyme may be further purified by conventional methods such as extraction, precipitation, chromatography, affinity chromatography, others like that.

However, it is an advantage of the invention that the culture medium with or without the host cells: ** ** can be used as such as an enzyme preparation without residual purification since endoglucanase fusion proteins can be secreted into the culture medium and exhibit activity in the culture medium consumed. ": Enzyme preparations are highly economical to obtain and use because specific isolation of the enzyme from the culture medium is unnecessary.

In addition to the endoglucanase fusion protein, the enzyme preparations may comprise one or more other enzymes which may be, for example, other cellulases, amylases, lipases, proteases, hemicellulases, xylanases: **, pectinases and / or oxidases, such as. Optionally, a second enzyme treatment may be carried out before, during or after treatment with the endoglucanase fusion protein, The enzyme treatment may, e.g., 35 comprise one or more amylase treatments (e.g., delimiter *), one or more cellulase treatments and / or one or more peroxidase and / or laccase treatments 14 What other enzymes are included in the enzyme preparation or used in the enzyme treatment depends on the application.

In addition to the fusion protein, the enzyme preparation may contain additives such as stabilizers, buffers, preservatives, surfactants and / or components of the culture medium. Preferred additives are those commonly used in enzyme preparations for an application in which the enzyme preparation is used.

Enzyme preparations may be provided as a liquid or as a solid, for example, as a dried powder or granules, particularly as a dust-free granule, or as a stabilized liquid. It is contemplated that the enzyme preparations may be further enriched or may be partially or completely removed from specific enzyme activities to satisfy the requirements of specific applications in various applications, e.g., the textile industry. A mixture of host-secreted enzyme activities may be advantageous in specific industrial applications, such as bio-finishing and bio-stone washing.

Endoglucanase fusion proteins and their preparations are useful, for example, in the textile, feed, vegetable oil, detergent and pulp and paper industries. They can be used to treat any cellulosic material, such as textile material, vegetable plants for animal feed. material for oil extraction or mechanical or chemical pulp from wood or recycled fiber. They may also be added to detergents, which usually contain excipients such as surfactants, surfactants, bleaching agents and / or fillers. In the present context, the cellulosic material refers to any material comprising cellulose or its derivatives as a significant component. The cellulosic material is contacted with an effective amount of the fusion protein under suitable conditions, such as at a suitable pH and temperature, and the reaction is allowed to continue, V, long enough for the enzymatic reaction to take place.

(30) Fusion enzymes are particularly useful in the treatment of textile materials, 7 such as fabrics and garments. The textile material may be made of natural cellulose-containing fibers or of artificial cellulose-containing fibers or of mixtures thereof with synthetic fibers. -, ···, is obtained from a mixture of fiber containing fibers. Preferably, the cellulose-containing materials are cotton, especially denim. "Denim" in the context of this invention refers to denim fabric, generally denim clothing, especially denim. 15 119434 denim is an indigo dyed denim, which can also be treated with indigo derivatives or indigo in combination with some other colors, for example, indigo dyed with a sulfur base.

Fusion endoglucanases are particularly useful in bio-5 stone washing and bio-finishing of textiles.

There are three steps to stone washing: removal of glue, rubbing and finishing. The first step, the glue removal process, is normally the first wet treatment of the jeans and involves the removal of starch or other glues commonly used on warp yarns to prevent their damage during the weaving process. Alpha-amylases are used to remove starch-based paste for improved and uniform wet handling. After removing the paste, the jeans are normally rinsed with water or transferred directly to the abrasion step.

The second step, abrasion, can be performed with enzymes or hoh-15 or both. In all cases, mechanical action is required to decolorize and treatment is generally performed on washing machines such as rum bean machines. The term "worn" refers to the appearance of the denim fabric when treated with cellulase enzymes or stones or both. Synonyms are "stone washed appearance" or "worn appearance". As a result of the removal of uneven color, there is between the dyed areas and the dyed areas. of contrasts.

··· * Abrasion is generally followed by a third step, a post-treatment which includes washing and rinsing steps during which detergents, optical brighteners, bleaching agents or emollients may be used. Enzymatic Concepts *: After 25 l the reaction must be stopped to prevent damage to the treated materials: · ': for example by inactivation of temperature and / or pH with the latter ··· comprising thorough rinsing and / or detergent washing. This ensures that the mechanical strength of the fiber is no longer compromised by the continuous presence of the enzyme (V).

30 As used in the present context, the term "biofuel washing" of a fabric or garment refers to the use of enzymes instead of pumice or to the treatment of a fabric or garment, in particular denim.

As noted above, treatment with cellulase can completely replace the treatment with pumice stones (for example, 1 kg of commercial enzyme equals 100 kg of stones). . ”1 35 However, screening glass treatment may also be combined with pumice stone treatment where: ** it is desirable to produce a highly worn finish. The peach-like effect of 16,119,434 creating a fine layer of protruding hair is also achieved by washing with a combination of neutral cellulase and pumice. The fusion proteins described are particularly useful for effectively providing a worn-out appearance and minimizing backstaining in biofuel washing.

Biofuel washing is typically carried out at a pH of about 3.0 to about 8.0 and preferably at a pH of about 5.0 to 7.0. The reaction temperature may range from about 30 to 80 ° C and is preferably from 50 to 60 ° C. The solution ratio (volume to volume ratio of liquid to fabric weight) may range from about 3: 1 to 20: 1, preferably from 5: 1 to 10: 1. The treatment time may range from 15 min to 90 min and preferably from 30 min to 60 min. It must be emphasized that the dose of 10 enzymes is highly dependent on the type of fabrics, machines, process conditions (pH, temperature, solution ratio, treatment time, denim load, process scale) and the type of enzyme preparation and the like. If so desired, pumice stones may be used in combination with endoglucanase fusion proteins. The required enzyme dose will then be significantly lower.

Appropriate doses and conditions can be determined by one of ordinary skill in the art.

The endoglucanase fusion proteins of the present invention provide unexpected benefits with high enzyme activity and low reflux, resulting in very good contrast. In addition, fusion proteins can be readily prepared and used over a relatively wide temperature range and pH range.

Further, endoglucanase fusion proteins are useful in the biofinishing of staples and clothing. '' Biofinishing 'refers to the use of enzymes in the controlled hydrolysis of cellulosic fibers to permanently modify the surface of the fabric or yarn, ,; * · 25 fabric feel, such as softness and smoothness, clarifies the surface structure by reducing lint, which results in color clarification, improves fabric: ***: settability, improves moisture absorption, which can also • improve color fastness.

Enzymatic anti-peeling can be carried out at any stage during the wet-treatment of the textile, preferably after optional stripping and / or bleaching, and conditions similar to those used in the washing of bio-stone can be used.

Endoglucanase fusion proteins with betaglucanase, hemicellulose, ·· *. glass or xylanase activity, are still useful for improving the quality of animal feed, · whereby the plant material is treated with enzymes.

Starch, proteins and fats can be readily digested in the digestive system of monogastric animals such as poultry and pigs, while most non-starch polysaccharides (NSPs), including, for example, barley and oat beta-glucans, due to the lack of such enzyme activities in the animal. In addition, the digestibility of other components, in particular animal fats, is reduced in the presence of NSP.

Beta-glucanases have been commercially used to alleviate the problems caused by barley and oat serum beta-glucans. 10 Beta-glucanases are known to reduce caused by soluble beta-glucan content of the intestinal viscosity and release plenty of glucans containing encapsulated cell walls of the nutrients. The use of betaglucanases improves animal productivity, which can be seen in improved weight gain and feed efficiency. Also, the incidence of sticky droppings in poultry la is typically reduced.

Steam pelletization is a major technology in feed processing throughout the world. Its advantages over the production of feed mashes include easier handling, reduction of toxic substances and organisms, and above all, improved feed efficiency. The heat-tolerance and high potency of the fusion proteins described herein make them suitable for feed applications.

The invention is illustrated by the following non-limiting examples. It should be understood, however, that the embodiments given in the above description and the prefixes are for illustrative purposes only and that various changes and modifications are possible within the scope of the invention.

··· · «* * · • ·» IT. Example 1. Production of EG28 cellulase • · '···' by Thermoascus aurantiacus ALK04242 in Trichoderma reese. . Basic methods of molecular biology were used for DNA (plasmids, *;] · * DNA fragments) isolation and enzyme treatments, E. coli transformations, etc. The basic methods used are described in the basic manuals of molecular biology, e.g. Sambrook et al (1989) and Sambrook and Russell (2001).

EG28 cellulase (SEQ ID NO: 2) encodes Thermoascus au-

• • I

The ce / 5A gene of ranf / äcus (SEQ ID NO: 1) was directly amplified by T by PCR.

aurantiacus from the genomic DNA of ALK04242 was isolated by the method of Raeder and Brodan, 35 (1985). Forward (SEQ ID NO: 9) and reverse primers (SEQ ID NO: 10) were designed based on published T. aurantiacus endoglucanase assay (AF487830). An amplified 1.3 kb product containing the exact gene (from the START codon to the STOP codon) was cloned as a SacII-Psfl fragment into the pBluescript II KS + vector. Two independent clones were sequenced and one clone was selected and named pALK1926: k $ i. The E. coli chicken-5 nanoparticle deposit number of pALK1926 in the culture collection of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH is DSM 17326.

The expression plasmid (pALK1930, Figure 1) was constructed for the production of recombinant T. aurantiacus cellulase EG28 / Cel5A (SEQ ID NO: 2). The ce / 5A gene, including its own signal sequence, was fused to exactly 10 T. raesei cb / 7 / promoter (cel7A) by PCR. The cb / 7f promoter, the cbh1 terminator, and the amdS marker gene were included as described by Paloheimo et al. 2003. The linear expression cassette (Figure 1) was isolated from the vector framework by restriction enzyme digestion, transformed into T. reesein A96, and transformants selected. acetamide as the sole nitrogen source. The host strain lacks four endogenous master cellulases: CBHI / Cel7A, CBHII / Cel6A, EGI / Cel7B and EGII / Cel5A. Transformations were performed according to Penttilä et al. (1987) with the modifications described in Karhunen et al. (1993). Transformants were purified on selection plates through individual conidia before being sprouted with PD.

Cellulase production of transformants was analyzed on culture supernatants of flask cultures (50 ml) grown in shakers. Transformants were grown for 7 days in complex M ·: V medium inducing the expression of cellulase (Joutsjoki et al. 1993) buffered with 5%! * ·· KH2PO4, pH 5.5. The enzyme activity of the recombinant protein was measured as: release of reduced sugars from culture supernatant from carboxymethylcellulose (2% CMC) at 50 ° C in 50 mM citrate buffer pH 4.8 · · · *. essentially as described in Bailey and Nevalainen 1981; Haakana et al., 2004.

..... Activity against barley betaglucan (1%) was also determined by measuring the release of reduced sugars at 50 ° C in 50 mM acetate buffer, pH 4.8 as described by Stälbrand et al., 1993. Recombinant protein production was also detected from the culture supernatant by SDS-polyacrylamide gel electrophoresis. Genotypes of selected transformants were analyzed by Southem blotting using an expression cassette as a probe.

I; *; ' The pH optimum of the heterologously produced Ta EG28 / Cel5A cellulase was determined in universal Mcllvane buffer at pH 4.0-8.0 using crude methoxymethylcellulose as substrate. As shown in Figure 2A, the pH optimum for EG28 / Cel5A cellulase activity is at pH 6.0. The optimum temperature for EG28 / Cel5A cellulase enzyme activity was determined to be 75 ° C (Figure 2B).

The selected transformant (RF6188) was cultured in a bioreactor to obtain material for application tests (see Example 4).

Example 2. Production of Recombinant Thermoascus aurantiacus ALK04242 EG28 + CtCBD Fusion Protein

To produce the recombinant Thermoascus aurantiacus EG28 + CtCBD fusion-10 protein (SEQ ID NO: 4), the CBHI / Cel7A cellulose binding cellulose (CBD) domain of Chaetomium thermophilum ALK04265 was ligated to EG28 / Cel5A cellulase. The construct contains the catalytic domain of EG28 (amino acids 1-334 of the full length polypeptide) linked to the C. thermophilumm CBHI / Cel7A CtCBD linker region and CBD (SEQ ID NO: 15).

Basic molecular biology techniques were used as described in Example 1. First, a unique SnaBI restriction site near the C-terminal end of the EG28 / Cel5A sequence was introduced by PCR. This allows direct fusion of any blunt-ended DNA after the amino acid Y334 of the EG28 / Cel5A polypeptide. C. thermophilumm CBHI / Cel7A linker and CBD region, 20 coding gene (cel7A, SEQ ID NO: 7) was amplified by PCR using for- * * ·; ·; ward (SEQ ID NO: 11) and reverse primers (SEQ ID NO: 12); and C. ther-: * V mophilum ALK04265 genomic DNA as a template. The amplified 1.6 kb · · product was ligated into the ce / 5A gene (after Y334) Ta ce / 5A-Ct ce / 7Alinker CBD j · ·; (SEQ ID NO: 3). The resulting plasmid was designated pALK1946.

The accession number of E. coli strain 25 containing pALK1946 in the culture collection of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH is DSM 18159.

The expression plasmid (pALK1948, Figure 1) for the construction of EG28 + CtCBD cellulase • · * M was constructed as described in Example 1. A linear 8.9 kb **; * '30 expression cassette (Figure 1) was isolated from the vector backbone / Vof1 restriction enzyme. · • * ·· digestion, transformed into T. reesei A33 (four strains encoding the major cellulases CBHI / Cel7A, CBHII / Cel6A, EGI / Cel7B and EGII / Cel5A) and transformants were selected as described in the example 1.

• ·

The pH and temperature optimum for the fusion protein were determined to be the same as for the wild-type EG28 protein.

20 119434

The selected transformant (RF6377) was cultured in a bioreactor to obtain material for application tests (see Examples 5-10).

Example 3. Production of Recombinant Acremonium thermophilum ALK04245 EG40 + CtCBD Fusion Protein To produce recombinant Acremonium thermophilum ALK04245 EG40 + CtCBD Fusion Protein (SEQ ID NO: 6) Cel7A domain. The construct contains the catalytic domain of EG40 cellulase (amino acids 1-234 of the full length polypeptide) linked to the CBHI / Cel7A linker region of C. thermophilum and CBD (CtCBD, SEQ ID NO: 15).

Basic molecular biology techniques are used as described in Example 1. The Acremonium thermophilum ce / 45A gene (SEQ ID NO: 8) is amplified by PCR using A. thermophilum ALK04245 genomic DNA using primers (SEQ ID NO: 13) and (SEQ ID NO: 13). no: 14). The 1.1 kb PCR fragment is cloned as a Sac11-Psfl fragment into the pBluescript II KS + vector. The unique A / rul restriction site near the C-terminal end of the EG40 sequence is then introduced by PCR. This allows direct fusion of any blunt-ended DNA after the amino acid S234 of the EG40 polypeptide. The Chaetomium thermophilum CBHI / Cel7A cellulase linker plus CBD region ···: '(ce / 7A) is amplified by PCR as described in Example 2 and its restriction fragment · · ·: V is ligated to the ce / 45A gene (After S234) At ce / 45A_Ct ce / 7Alinker CBD (SEQ ID NO: 5). Expression Plasmid et al: *: EG40 + CtCBD for cellulase production is constructed and recombinant protein: 25 (SEQ ID NO: 6) is produced in Trichoderma as described in Example 1.

Example 4. Efficacy of EG28 Cellulase in Denim Finishing at Different Temperatures [*] EG28 cellulase of Thermoascus aurantiacus produced * ··· 1 'in Trichoderm ( strain RF6188), as described in Example 1, was tested for its ability to create a similar worn appearance obtained with *; ***: pumice in denim biofilm washing at various temperatures. A potent denim finishing enzyme, a commercially available EGII-enriched acidic cellulase preparation, produced in Trichoderma (US 5,874,293), was used as a reference.

: J Indigo-stained denim twill jeans were used as a test medium after the removal of the adhesive with ECOSTONE® A200 alpha-amylase. The pulp 21 119434 glass treatments were performed on an Electrolux Wascator FOM 71 CLS washing machine under the conditions described in Table 1.

The endoglycanase activity (ECU) of the enzyme preparations used was measured as the release of reduced sugars from hydroxyethylcellulose 5 as previously described (Bailey and Nevalainen, 1981). EGII-enriched enzyme concentrate and EG28 were dosed at 220 ECU / g and about 380 ECU / g of fabric, respectively. The enzymes were tested at their optimum pH range, EGII preparation at pH 5 and EG28 preparation at pH 6. After removal of water, the enzyme was inactivated (for 10 minutes at 40 ° C) by raising the pH above 11 with 10 sodium hydroxide. The jeans were then rinsed three times with water and dried in a tumble dryer.

The bio-stone wash effect / abrasion level was evaluated by measuring the color reflectance values on a Minolta CM 2500 spectrophotometer using L * a * b * color space coordinates (light type D65 / 20). The color of the right and wrong sides of the denim and the color of the pockets were measured after the removal of the glue (i.e. before the cellulase treatment) and after the cellulase treatment. Each measured value from the right side, the jerk side, or the pockets, was approximately the average of 40, 20, or 12 measurements, respectively. Two pairs of jeans were used in each test and the final result was their mean. The results are shown in Table 2 and Figure 3.

20 · • »» »» »* * * ♦ ♦ ♦ ♦ ♦ · ♦ · · · · • »· · * • · 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 · • · · • • • «22 119434

Table 1. Test conditions / process parameters used in cellulase treatments

Prosessiparametri________

Denim load__1.3 kg ____

Water 19 liters_ pH control (pH 5-5.3) 35 g Na2HP042H2O + 22 g citric acid pH controlHi (pH 6.1-6.3) 35.5 g Na2HPQ42H2Q + 15 g citric acid

Time__45 min_ Temperature__40, 50, 60 or 70 ° C_

Cellulose dose__220 or about 380 ECU / g fabric_

Table 2. Color measurements of denim treated with EG28 cellulase at various temperatures Treatment with EGII-enriched preparation was used as a reference. L * indicates brightness, -b * is blue, + b * is yellow. __

Enzyme - ECU / g Conditions Before Cellulase · Preparation of cellulose deltaL * deltab * after clothing treatment ____ r ______ £ ___

Right side: ____________ EG28 380 70 ° C, pH 6.1-6.3 23.67 -16.07 32.63 -16.94 8.96 -0.87 • _ “EG28__370 60 ° C, pH 6.1-6.3 23 , 67 -16.38 31.94 -17.09 8.27 -0.71 Γ **: EG28 380 50 ° C, pH 6.1-6.3 23.62 -16.24 31.91 -16 , 86 8.30 -0.63 S Λ ~ ------- ". *.: EG28 380 40 ° C. PH 6.1-6.3 23.75 -16.07 30.57 -17.32 8.82 -1, 25 • ·· "'r" r ............ - -:. *! EGII Enriched 220 60 ° C pH 5.2-5.3 23.45-16, 10 32.90 -17.29 9.46 -1.19 • · · "* / Downside: _______________ • · *" "* · ί · * EG28 380 70“ C, pH 6.1-6.3 49 , 76 -6.95 49.80 -10.43 -0.18 -3.48 ----- -—--- _ '* · ............. ”EG28 370 60 ° C · PH 6.1-6.3 49.55 -6.87 50.78 -9.57__1.24 -2.71 EG28__380 50 ° C, pH 6.1-6.3 49.92 -7.05 50.49 -9.83__0.57 -2.78 J '· EG28__380 40 ° C, pH 6.1-6.3 49.89 -6.93 49.73 -9.73 __- 0.16 -2.80. · ··. EGII Enriched 220 60 ° C, pH 5.2-6.3 49.75 -6.82 48.28 -12.28 -1.47 -5.45

Pockets: _________ iti: EG28__380 70 ° C, pH 6.1-6.3 77.40 -8.14 68.53 -12.60 -8.86 -4.47-1; EG28 374 60 ° C, pH 6.1-6.3 77.91 -7.71 70.29 -12.34 -7.62 -4.63 •> 1 '', *. EG28__380 50 ° C, pH 6.1-6.3 78.11 -7.62 70.63 -12.02 -7.49 -4.40 • 1 * ··· EG28 380 40 ° C, pH 6.1-6.3 77 , 51 -7.60 70.47 -11.91 -7.04 -4.32 ··· ^ .....

EGII Enriched 220 60 ° C, pH 5.2-5.3 77.46 -7.68 66.18 -14.17 -11.29 -6.49 23 119434

The results in Table 2 and Figure 3 show that the best rubbing effect with EG28 (strain RF6188) was obtained between 50 and 70 ° C. Using a dose of 380 ECU / g fabric at 70 ° C, EG28 produced a similar level of abrasion (L *) as using a dose of 5,220 ECU / g fabric at 60 ° C. However, the effect of backstaining (redeposition of indigo color) on the wrong side of the denim and pockets was lower with EG28 (higher brightness, less blue) than with EGII enriched. Also the contrast on the right side of the denim looked better.

Example 5. Efficacy of EG28 + CtCBD Fusion Protein over EG28 Cellulase in Denim Finishing

The recombinant Thermoascus aurantiacus EG28 + CtCBD fusion protein produced in Trichoderma (strain RF6377) as described in Example 2 was compared to EG28 from strain RF6188 in a denim biofilm-15 wash at pH 6.60. The denim and the test system for biofuel washing were similar to Example 4 except that the amount of denim was equalized to 1430 g with an additional piece of different denim which was not included in the measurements. The effect of the cellulase treatment was evaluated as in Example 4.

The results in Table 3 show that the bio-20 stone washing effect of EG28 + CtCBD was very good at the low dose. For strain RF6377, the same level of abrasion (brightness L *) was obtained 6.5 times lower dose relative to RF6188. The incorporation of the cellulose binding domain (CBD) into EG28 cellulase did not increase the undesirable back dyeing effect but greatly increased the desired washing performance.

· »* * • M« · · · • M «··· • t • * · · · · · · · · · · · ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Table 3. Color measurements of denim handwoven from EG28 and EG28 + CtCBD cellulases at 60 ° C 24 119434 L * indicates brightness, -b * is blue, + b * is yellow; _ ^ __

Enzyme-ECU / g Before cellulase- Cellulase- DeltaL * deltab * Preparation of clothing after treatment ___ L * b * L * b * ___

Right: EG28 325 23.40 -16.02 31.01 -17.12 7.62 -1.10 EG28 + CtCBD 50__23.81 -16.21 31.60 -16.92 7.80 -0.71

Downside: ___ _ EG28 325 49.46 -6.83 50.03 -9.62 0.57 -2.79 EG28 + CtCBD 50 49.36 -7.03 50.62 -9.61 1.27 - 2.58

Pockets: ________ EG28__325 76.11 -8.36 68.53 -12.46 -7.58 -4.10 EG28 + CtCBD 50 76.02 -8.45 68.81 -12.39 -7.21 -3 , 94 5

Example 6. Efficacy of EG28 + CtCBD Cellulase as compared to EGII Cellulase in Denim Finishing

The recombinant Thermoascus aurantiacus EG28 + CtCBD fusion protein produced in Trichoderma (strain RF6377) was compared to a commercially available EGII-enriched acidic cellulase preparation (US 5 874 293) in a biofilm wash.

: * V The test system for washing stoneware was similar to Example 4 except that several pieces of different denim types (legs) from Ukos Sport (Belgium) and Vicunha (Brazil) were used (1.2 kg in total). The conditions for EG28 + CtCBD and 15 EGII treatments were similar to Table 4. The effect of the cellulase treatment was evaluated as in Example 4.

The results of the experiments on four different denim fabrics are reported as - / *, as shown in Tables 4 and 5. Using the enzyme dose *; *. * Of EG28 + CtCBD, a similar brightness level was obtained as using EGII- * ·; ** 20 a dose of enriched preparation of 1000 ECU / g of fabric. The potency of EG28 + CtCBD was roughly only half that of the ECU activity compared to EGII-enriched cellulase. Also, the culture medium of EG28 + CtCBD produced by the recombinant host is about twice as high in volume as re- 25,119,434 EGII medium produced by a combinatorial host in a stonewashing. Significantly less back staining was observed on the downside of the denim when EG28 + CtCBD was used. The efficacy of EG28 + CtCBD at 50 ° C was better at pH 6 than at pH 5.

• · 1 · i1 · • v · • »1 • • • • • 1« '· • • • # 1 • • f1 · • tl • ♦ · * »1

Ml t · «·« ·· M • ·

• M

· · · · · · · · · · · · · · · · · · · · · · · · · · · ···

I I I «·· I

• · t • 9 • ·

• M

Table 4. Color measurements with EG28 + CtCBD cellulase at pH 5 and 6 on the right side of the hand denim 26 119434 Treatment with EGII-enriched preparation was used for comparison. L * il-_is brightness, -b * is blue, + b * is yellow .__

Enzyme-Denim ECU / g Oval ratios Before cellulase- Cellulase deltaL * deltab * Preparation of garment after treatment After ________ L * b * __ L »b * _______

Atlanta, 19.96 -15.91 27.44 -18.49 7.48 -2.58

Smart sports

Mathew, pH adjusted 18.28 ~ 7'97 21.87 -11.71 3.59 '3-74

Ukos sport,. „--- from acetic acid -------—.........

Nostalgy,. 19.12 -8.78 23.90 -13.50 4.78 -4.72 55 min, EGII- Ukos sport 1000 50 ° C, pH 5 ____- ___ enriched Viounha, 17.24 -10.49 21.51 -14 , 93 4.27 -4.44

Savannah _____

Average 18.65 -10.79 23.68 -14.66 5.03 -3.87 __value__ _____

Atlanta, 19.79 -16.02 25.88 -18.44 6.09 -2.42

Smart sports ___

Mathew, 18.07 -8.58 21.45 -11.80 3.38 -3.22

Thunderstorm sport pH adjusted _ _ _

Nostalgy, with acetic acid 19.05 -8.73 22.65 -12.69 3.60 -3.96 ♦ · ·

Ukos sport 55 min, _ • * I EG28 + CtCBD Vicunha, 500 50 ° C, pH 5 17 38 -10.11 20.75 -14.18 3.37 -4.07 · * · · Savannah ♦ ^ S »- _ ______ ____:. ·, Central 18.57 -10.86 22.68 -14.28 4.11 -3.42 * * ·

East ·. value • · * —-------- * ··· * Atlanta, 19.98 -16.18 27.35 -18.20 7.37 -2.02 ···

Smart sports _____

Mathew, 17.51 -8.84 21.40 -11.62 3.89 -2.78 ", Ukos sport pH adjusted • ·· · —---------------- - * ··· Nostalgy, with Acetic Acid 18.58 -8.65 22.96 -12.75 4.38 -4.10 f 8 '* · Musky Sport 55 min, ______ EG2 $ + CtCBD Vicunha, 500 50 ° C, pH 6 17.50 -10.16 21.49 -14.06 3.99 -3.90 • ', ***. Savannah • ·, ------ - —— —— - --- - '· * Average 18.39 -10.96 23.30 -14.16 4.91 -3.20 Φ · * · · s value

• · · __. _ .._________ I

• »-——— 1 ♦ · · * ·

Table 5. Color measurements with EG28 + CtCBD cellulase at pH 5 and 6 on reverse side of hand-denim 119434 27 Treatment with EGII-enriched preparation was used for comparison. L * il-_lays brightness, -b * is blue direction __

Enzyme-Denim ECU / g Oval ratios Before cellulase- Cellulase-deltaL * deltab * preparation for garment treatment after treatment __________ L * b * L * __ b ^ _______

Atlanta, 47.98 -7.25 40.18 -14.63 -7.80 -7.38

Smart sports

Mathew, pH adjusted 38 · 41 –7 · 17 36 · 11 ’10 · 93’ 2 · 30 ”3 · 76

Lightning sport with acetic acid EGII-1000 ""

Nostalgy, 55 min, 46.70 -6.06 41.87 -11.73 -4.83 -5.67 enriched

Ukos sport 50 ° C, pH 5

Vicunha, 35.65 -9.24 33.48 -12.44 -2.17 -3.20

Savannah__Average Value ____ 42.19 -7.43 37.91 -12.43 -4.28 -5.00

Atlanta, 48.01 -7.29 41.82 -13.25 -6.19 -5.96

Smart sports ____

Mathew, 37.60 -7.27 35.59 -10.50 -2.01 - 3.23 pH adjusted

Ukos sport - with acetic acid ------ EG28 + CtCBD Nostalgy, 500 „. 46.79 -5.99 44.75 -9.37 -2.04 -3.38 φ · | 55 min,? V Utl0-i-S-L0? - 50 ° C, pH 5 ------- ----! * .ί Vicunha, 35.08 -9.48 33.72 -11.80 -1.36 -2.32 * · • ·

Savana ·· * # - —-- • · * · Average 41.87 -7.51 38.97 -11.23 -2.90 -3.72 ··· ---- ”---- ---------- -. · **, Atlanta, 47.55 -7.43 42.41 -13.30 -5.14 -5.87 'M »

Ukos sport ___ ··, Mathew, 37.27 -7.58 36.01 -10.27 -1.26 -2.69 i ** pH adjusted> ·· Ukos sport •; - with acetic acid -----— - · ** EG28 + CtCBD Nostalgy, 500 rl_. 47.02 -6.01 44.61 -10.13 -2.41 -4.12 ,,, 55 min,! * ?! Uk0SSpOrt 50 ° C, pH 5 _____—- • t >>! Vicunha, 34.84 -9.39 33.53 -11.20 -1.31 -1.81 •, ·, Savannah • · · ---- - - - "-

Average Value 41.67 -7.60 39.14 -11.23 -2.53 -3.62 • * ·· * 28,119434

Example 7. Effect of pH on the Effect of EG28 + CtCBD Cellulase Preparation at 50 ° C and 60 ° C

The recombinant Thermoascus aurantiacus EG28 + CBD fusion protein produced in Trichoderma (strain RF6377) was tested for its potency 5 in denim biofilm washing at various pHs at 50 ° C and 60 ° C.

Indigo-dyed denim twill (different from previous examples), which had been de-glued and cut into pieces, was used as test material. Cellulase treatments were performed on an LP-2 scrubber as follows. Approximately 7.2 g of denim fabric (about 12 cm x 12 cm strip), 200 ml of Mclwane sit-10 phosphate buffer and 90 steel balls (0.6 cm diameter) were loaded into 1.2 liter containers. The washer was operated for 120 minutes at various pHs 4-8 using temperatures of 50 ° C and 60 ° C. After removing the strips from the tanks, they were rinsed with water and soaked in NaOH-containing water (pH> 11) with stirring for 10 minutes. The strips were then soaked in warm water containing liquid detergent (OMO 15 Color) and stirred for 10 minutes, followed by gentle rinsing with warm water several times. The strips were dried at room temperature. The bio-stone wash effect was evaluated by measuring the color in Reflection Units as described in Example 4. Each measured value on the right side of the de-nameplate was an average of at least 20 measurements.

The results shown in Tables 6 and 7 and Figures 4 and 5 show that,. the optimum pH range for EG28 + CtCBD cellulase at 50 ° C is 5.5-6.5 and at 60 ° C 5-7.

i · • «• • • * * * * li li li li li li li

IM lii «I

• I Ml «·

• I

• ·· • · S • · • «··· *« · • · I I «··· • · ·

IM

• · • · ·

• I I IM I

Ml • «• ·

Ml 29 119434

Table 6. Color measurements on EG28 + CtCBD cellulase at different pHs on the right side of the denim treated at 50 ° C in a washing machine _L1 indicates brightness, -b1 indicates a blue direction__

Enzyme - ECU / g Conditions Before Cellulase - Preparation of Cellulase L1 Treatment for Clothing Treatment__After Addition _____L «b» __ L1____

Buffer Control__0__pH 6 17.28 -13.07 18.77 -14.13 1.49

Buffer Control__0 pH 8 17.01 -13.4 18.18 -14.45 1.17 EG28 + CtCBD 1000__pH 4 16.79 -13.59 20.86 -16.40 4.07 EG28 + CtCBD 1000__pH 5 17.34 -13.18 21.59 -16.44 4.25 EG28 + CtCBD 1000 pH 5.5 17.30 -13.25 22.06 -16.39 4.76 EG28 + CtCBD 1000__pH 6 17.35 -13, 42 22.29 -16.47 4.94 EG28 + CtCBD__1000__pH 6.5 17.31 -13.41 22.48 -16.65 5.17 EG28 + CtCBD 1000 pH 7 17.34 -13.25 21.55 -16.23 4.21 EG28 + CtCBD 1000 pH 8 16.91 -13.63 19.62 -15.72 2.71 5 Table 7. Color measurements with EG28 + CtCBD- $ ellulase at various pH values in a washing machine at 60 ° C: on the right side of denim treated with L1 indicates lightness, -b1 is blue direction__ (1 (Enzyme-ECU / g Conditions Before Cellulase- Cellulase Treatment L1! 2 · Preparation after Clothing Addition • · · I j \ \ L1 b1 L1 b1 / / EG28 + CtCBD 1000__pH 4 17.19 -13.58 20.48 -15.77 3.29! · Ϊ 1 EG28 + CtCBD 1000 pH 5 17.10 -13.24 22.74 -16.59 5.64 • 111 ———! .Ϊ: EG28 + CtCBD 1000 pH 5.5 17.10 -13.54 22.66 -16.61 5.56 • • e ----— - 1 ----- ----— —— ! ...: EG28 + CtCBD 1000__pH6__17.10 -13.65 22.83 -16.74 5.73 EG28 + CtCBD 1000__pH 6.5 17.19 -13.47 22.65 -11.72 5.46 j2 .. EG28 + CtCBD__1000__pH7__16.99 -13.72 22.10 -16.66 5.11 i "1i EG28 + CtCBD 1000 pH 8 17.13 -13.68 19.35 -15.15 2.22 ··· ————- - - -------: -

Mt ··. Buffer Control 0 pH 6 17.36 -13.6 18.95 -14.28 1.59 »·» • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · at 30 119434

Example 8. Efficacy of EG28 + CtCBD Cellulase Preparation at Denim Finishing at Different Temperatures

The recombinant Thermoascus aurantiacus EG28 + CtCBD fusion protein produced in Trichoderma (strain RF6377), as described in Example 5, Example 1, was tested for its ability to create a similar worn appearance obtained by pumice stone washing at different temperatures. A potent denim finishing enzyme, a commercial EGII-enriched acidic cellulase preparation (US 5,874,293) and a neutral cellulase preparation ECOS-TONE® 01 were used as reference.

The test system for washing stoneware was similar to Example 4 except that different denim wools and a washing time of 55 minutes were used. The enzyme activity (ECU) of the EG28 + CtCBD enzyme preparation and the EGII-enriched enzyme preparation was measured as in Example 4. The activity of the neutral cellulase preparation ECOSTONE® C1 (Bailey and Nevalainen, 1981; Haakana et al., 2004). ECOSTONE® C1, EG28 + CtCBD or EGII-enriched formulations were dosed at 250 NCU / g, 500 ECU / g or 1000 ECU / g of fabric, respectively. The effect of the cellulase treatment was evaluated as in Example 4.

The efficacy of EG28 + CtCBD at temperatures of 40-70 ° C, pH 6 is shown in Table 2. in lock 8 and figure 6. The preferred temperature range for the enzyme is 50-70 ° C. Low temperatures, such as 40 ° C, can be used to obtain an abrasion level with less discoloration if a darker appearance is desirable.

The results are also in agreement with the results obtained in Example 6: where the efficacy of EG28 + CtCBD in biolithic washing was shown to be about: 3: twice as effective as the commercial EGII-enriched acidic cellulose preparation. Acidic salivaases generally have a tendency to repel. Neutral cellulases such as ECOSTONE® C1 typically cause ···. 30 ways of lower back coloring therefore leading to good contrast. The results obtained here · · * · 1 show that a similar effect was obtained with EG28 + CtCBD- ··· * · 1 1 cellulase and neutral salivases (Table 8).

• · · • • • · · · · * 1 ® · · · · 2 • · 3 · 1 »31 119434

Table 8. Color measurements of denim treated with EG28 + CtCBD cellulase Treatment with EGII-enriched acidic cellulase and neutral ECOSTONE® C1 cellulase preparation was used as a reference. L * indicates 5 brightnesses, -b * is blue, + b * is yellow. __

Enzyme- Active- Conditions before salivase- Cellulase treatment deltaL * deltab * preparation / g after treatment __clothing L * b * L * b *

Right side: EG28 + CBD__500 ECU / g at 70 ° C, pH 6.2 17.38 -13.49 27.42 -17.27 10.05 -3.78 EG28 + CBD__500 ECU / g at 60 ° C, pH 6 , 2 16.76 -13.31 26.01 -17.49 9.25 -4.18 EG28 + CBD__500 ECU / g at 50 ° C, pH 6.2 16.78 -13.40 25.00 -17, 44 8.22 -4.04 EG28 + CBD__500 ECU / g at 40 ° C, pH 6.2 16.76 -13.21 22.86 -17.25 6.11 -4.04 EGII-enriched 1000 ECU / g 60 ° C, pH 5.1 17.24 -13.38 26.59 -17.64 9.35 -4.26 ECOSTONE C1 250 ECU / g 60 ° C, pH 6.5 17.38 -13.53 26.91 -16.98 9.53 -3.45

Downside: EG28 + CBD__500 ECU / g at 70 ° C, pH 6.2 45.67 -6.33 41.78 -13.83 -3.90 -7.51 EG28 + CBD__500 ECU / g at 60 ° C, pH 6.2 45.60 -5.95 42.72 -13.05 -2.88 -7.10 EG28 + CBD__500 ECU / g at 50 ° C, pH 6.2 45.67 -5.92 41.84 - 13.35 -3.86 -7.44 EG28 + CBD__500 ECU / g at 40 ° C, pH 6.2 45.80 -5.72 41.66 -12.52 -4.14 -6.81 ··· EGII-enriched 1000 ECU / g at 60 ° C, pH 5.1 46.10 -6.19 39.89 -15.24 -8.21 -9.06 * · e * ~~ i. - -.

ECOSTONE C1 250 ECU / g 60 ° C, pH 6.5 45.63 -6.46 42.49 -12.76 -3.14 -6.31 • · i - I »i ·! _ II _____ • · • · Example 9: Effect of EG28 and EG28 + CtCBD Cellulases on Biofinishing (Anti-Puffle) · »· EG28 Cellulase Strain RF6188 and EG28 + CtCBD Strain RF6377 on Cotton in the prevention of bumping of knitwear j * ·. tested. Cellulase treatments were performed on an Electrolux Wascator FOM 71 CLS washing machine under the conditions described in Table 9.

· «· (.J. 'Low-quality polo-collared knit shirts;!. * Surface, two types of pieces made of 100% jersey-based cotton • · **; · * 15 fabrics or elasticated 95% cotton and 5% lycra were used as test material with filler, samples were first pre-washed for 10 minutes at 60 ° C with 1 ml / L surfactants / wetting agents (Sandoclean PCJ from Sandos and Imacol CN Clariant) and rinsed three times with liquid. Then, the cotton needles were then treated with cellulase at 60 ° C for 60 minutes in the presence of the same textile auxiliaries used for prewash, and the enzyme was inactivated as described in Example 4, except at 60 ° C during alkaline rinsing. rinsed three times and then dried in a tumble dryer.

Table 9. Test conditions / process parameters used in bio finishing treatments

Prosessiparametri__

Fabric load__1.0 kg_

Water__15 liters_

Sandoclean PCJ and Imacol CN 1 ml / L

Buffer / pH control (pH 5-5.3) about 3 ml acetic acid (80%)

Time__60 min_ Temperature__60 ° C_

Cellulose dose_ 1200 ECU / g cloth_

The effect of cellulase treatment was assessed visually with the naked eye 10 and the magnifying glass. The pre-washed sample without enzyme was used as a control.

The results show that EG28 + CtCBD improved anti-numbness properties. The linen surface of the knitwear was noticeable ···! significantly reduced after treatment with EG28 + CtCBD compared to the · · ·: V control (treatment without enzyme). EG28 also had an anti-puncture effect.

Example 10: Stability test of feed pelletization • · »

* ··. Two cellulose preparations, EG28 (strain RF6188) and EG28 + QCBD

Φ (strain RF6377) was tested separately in an experiment simulating an industrial feed production process. Before testing, the spray-dried EG28 or EG28 + CtCBD formulations were milled with wheat flour to improve homogeneity at the re- * ··· * level. EG28 and EG28 + CtCBD enzymes premixed with flour were added at 200 g / t and 500 g / t feed, respectively. Enzyme m. · **. an overdose was used to facilitate analysis at high temperatures in which ·· *. *. activity may have been substantially reduced in the pelleted samples.

• «·:; j .: 25 The mill and blender are used to produce flour mixes as part of a · · * ··· * semi-industrial feed equipment with a nominal pelletizing capacity of 5 t / h.

33 119434

The mill is a Champion hammer mill equipped with 0 3.0 mm die. The ground raw material is mixed in a 2500 liter horizontal mixer at 27 rpm before being transferred to a miniature feed mill. The equipment comprises a horizontal mixer (volume 700 L, speed 48 rpm, 5 300 kg mixing batches), Skjold TR dosing screw and Kahl cascade mixer (length 130 cm, diameter 30 cm, speed 155 rpm, equipped with 37 adjustable pallets). The compression time for 300 kg / h is approximately 30 seconds. Installed on the side of the cascade mixer is a manifold with a water flow and 3 steam valves from which the steam is fed to the flour. The steam is generated in a high pressure 10 steam boiler with a maximum capacity of 400 kg steam / h. The tests are conducted at 2 atmospheric overpressure and steam is passed through a pressure relief valve that controls the addition of steam to the cascade mixer. Three valves in the manifold are used to fine-tune the desired flour temperature. The temperature of the flour was measured with a digital thermometer inserted into the outlet of the cascade mixer just before the flour entered the pellet die. The pellet press is Simon Heesen, type labor (monoroll) with 0 3 mm x 35 mm nozzle and 7.5 kW motor. Inner diameter of the die: 173 mm, height of the press roll: 50 mm, diameter of the press roll: 140 mm, speed 500 rpm and nominal capacity: 300 kg / h. Samples were taken after pellet pressing and cooled in a fractional-bottomed cooling box with a fan: 1500 m3 air / h.

A 300 kg batch of wheat based feed paste was produced. The premix was produced from 10 kg of this flour and 200 g (or 500 g) of the test enzyme in a 70-liter forced mixer. Mixer speed: 45 rpm. Mixing time: 10 min. This • · \ * ·: premix was added with 290 kg of feed to a horizontal mixer in a low-feed mill and stirred for 10 minutes. After sampling the mash, the feed was pelleted through a pellet press (die 0 3 mm). Already: ** ·; ho was heated to the target temperature (65-90 ° C) by adjusting the steam addition * * * to the cascade mixer. For each temperature, a sample was first taken 10 minutes after: · reaching the target pelletization temperature (measured from the flour just before the pelletizing). Samples were taken over 1.0 minutes, corresponding to 5.0 kg of pelleted feed. The sample was taken in approximately 500 gram sub-samples and placed in a cooling box for 10-15 seconds after the pellets had left the pellet press. All samples were ventilated and cooled at room temperature for 15 minutes. were homogeneously subdivided into · · · 35 sand sampler and filled into plastic bags.

Samples were analyzed as follows: 2.5 g of a well-ground sample and 20 ml of acetate buffer (0.05 M, pH 5.0) were stirred for 30 minutes at room temperature, centrifuged (10 min, 400 rpm) and diluted. 1.0 ml of the clarified sample in triplicate was equilibrated for 5 minutes in a water bath at 40 ° C. The reaction was started with an additional one of Beta-Glucazyme tablet (Megazyme, Ireland) without stirring. After exactly 30 minutes, the reaction was quenched by the addition of 5.0 mL of Trizma Base 1% (w / v) (Sigma-Aldrich) with vigorous stirring with a vortex mixer. The tube was left at room temperature for about 5 minutes; the slurry was mixed again and filtered through Whatman 1 filter paper. The absorbance of the filtrate was measured at 590 nm. The results were analyzed using a standard curve.

The recovery of enzyme activity before and after pelleting is shown in Table 10. The effect of the reflection temperature on the recovery of betaglucanase activity is also shown in Figures 7 and 8. The EG28 and EG28 + CtCBD-15 cellulases were stable up to 80 ° C. EG28 + CtCBD cellulase tended to be more stable than EG28 at 90 ° C pelletization temperature.

Table 10. Recovery of enzyme activities before and after pelleting (flour horizontally from a mixer) at temperatures ranging from 65 ° C to 90 ° C

Pelleting Temperature _Betaglucanase Inactivity (FBU / kg Feed) _..li * __ (EG28) _ (EG28 + CtCBD) _ • V Flour from Horizontal 239,000 ± 9,000 (100%) 1,148,000 ± 48,000 (100%) • «____________-. _. . . --------e: ': Pellet (65 ° C) _ 227,000 ± 16,000 (95%) 1,007,000 ± 67,000 (88%): Pellet (70 ° C) __ 217,000 ± 5,000 (91%) 1012,000 ± 54,000 (88%). Pellet (75 ° C) __ 227,000 ± 14,000 (95%) 993,000 ± 49,000 (86%)

Pellet (80 ° C) 215,000 ± 6,000 (90%) 1030,000 ± 87,000 (90%)

Pellet (85 ° C) 188,000 ± 7,000 (79%) 904,000 ± 56,000 (79%) \ ... Pellet (90 ° C) 149,000 ± 8,200 (62%) 861,000 ± 32,000 (75) %) e · X r .......

··· AA

. 20 ··· • · · · · · · · · · · · · · · · · · · · · · · · ···

List of deposited organisms

Stock Included Deposit institution Deposit date Deposit number __plasmldi____

Acremonium - CBS (1) September 20, 2004 CBS 116240 thermophilum ALKQ4245________

Thermoascus - CBS (1) September 20, 2004 CBS 116239 Aurantiacus ALKQ4242_____

Chaetomium - CBS (2) November 8, 1995 CBS 730.95 thermophilum ALKQ4265_____

Escherichia coH pALK1946 DSMZ * 3 * April 7, 2006 DSM 18159

Escherichia coli pALK1926__DSMZ__13. May 2005 DSM 17326 (1) Centralbureau Voor Schimmelcultures at Uppsalaal 8, 3584 CT, Ultrecht, The Netherlands (2) Centralbureau Voor Schimmelcultures at Oosterstraat 1, 3742 SK BAARN, 5 The Netherlands (3) Deutsche Sammlung von Microorganismen und Zellkulturen GmbH (DSMZ), Mascheroder Weg 1 b, D-38124 Braunschweig, Germany • * e ** a * • e · • ee • ea · aaaae • aa * aeaaaaaaa aaa aa aaa aaa aaa aaa aaa aaa aaa aaa a aa * aaaa aaa aaa aaa aaa aaa a aa * aaa * aa * 119434

sekluetxt.txt SEQUENCE LISTING

<110> AB Enzymes Oy <120> Enzyme Fusion Proteins and their use <130> 2060570 <160> 15 <170> Patent Version 3.2 <210> 1 <211> 1317

<212> DNA

<213> Thermoascus aurantiacus <220> <221> gene <222> Cl) .. (1317) <220> <221> Intron <222> ¢ 107) .. (161) <220> <221> Intron <222> (221) .. (280) <220> <221> Intron <222> (434) .. (492) <220> <221> Intron <222> (558) .. (631) <220> <221> intron <222> (730) .. (790) ..ΙΓ <400> 1 j ·. ·, atgaagctcg gctctctcgt gctcgctctc agcgcagcta ggcttacact gtcggcccct 60 • · φ ·

. ·. i ctcgcagaca ggaagcagga gaccaagcgt gcgaaagtat tccaatgttc gtaacatcca 120 * · M

:. ·. cgtctggctt gctggcttac tggcaactga caatggcgaa gggttcggtt caaacgagtc 180 • · · Φ · ·. cggtgctgaa ttcggaagcc agaaccttcc aggagtcgag gtcagcatgc ctgtactctc 240 • * »···. * ··. tgcattatat taatatctca agaggcttac tctttcgcag ggaaaggatt atatatggcc 300 • tgatcccaac accattgaca cattgatcag caaggggatg aacatctttc gtgtcccctt 360. . ·. tatgatggag agattggttc ccaactcaat gaccggctct ccggatccga actacctggc 420 • · · * ·. ···. agatctcata gcggtacatt tcaattccac catgtttgga gctgtcttcg ttgtgctgac 480 • · ··· *. atttaatggt agactgtaaa tgcaatcacc cagaaaggtg cctacgccgt cgtcgatcct 540 · · · · * "cataactacg gcagatagtg aggtccccgg ttctggtatt gctgctgtat atctaagtag 600 t ·" * "* atatgtgttt ctaacatttc cacgatttca gctacaattc tataatctcg agcccttccg 660 ·· • · *.! atttccagac cttctggaaa acggtcgcct cacagtttgc ttcgaatcca ctggtcatct 720 t tcgacactag taagctgaac acccgaaatt aactgagtct gagcatgtct gacaagacga 780 119 434 page 1 sekluetxt.txt tccatgaaag ataacgaata ccacgatatg gaccagacct tagtcctcaa tctcaaccag 840 gccgctatcg acggcatccg ttccgccgga gccacttccc agtacatctt tgtcgagggc 900 aattcgtgga ccggggcatg gacctggacg aacgtgaacg ataacatgaa aagcctgacc 960 gacccatctg acaagatcat atacgagatg caccagtacc tggactctga cggatccggg 1020 acatcagcga cctgcgtatc ttcgaccatc ggtcaagagc gaatcaccag cgcaacgcaa 1080 tggctcaggg ccaacgggaa gaagggcatc atcggcgagt ttgcgggcgg agccaacgac 1140 gtctgcgaga cggccatcac gggcatgctg gactacatgg cccagaacac ggacgtctgg 1200 actggcgcca tctggtgggc ggccgggccg tggtggggag actacatatt ctccatggag 1260 ccggcgg cgta tcagcagata cttcctattt tgactccgta tctttga 1317 <210> 2 <211> 335

<212> PRT

<213> Thermoascus aurantiacus <400> 2

Met Lys Leu Gly ser Leu val Leu Ala Leu ser Ala Ala Arg Leu Thr 15 10 15

Leu ser Ala Pro Leu Ala Asp Arg Lys Gin Glu Thr Lys Arg Ala Lys 20 25 30 h Phe Gin Trp Phe Gly ser Asn Glu ser Gly Ala Glu Phe Gly Ser 35 40 45

Gin Asn Leu Pro Gly Val Glu Gly Lys Asp Tyr lie Trp Pro Asp Pro 50 55 60 * ♦ · · * ·· Asn Thr lie Asp Thr Leu lie Ser Lys Gly Met Asn lie Phe Arg val:: 65 70 75 80 • · • ·

• »I

*. . ρΓΟ pfog Met Met G- | U Apg Leu Va1 ρΓΟ Asn Ser Met G1y Ser ρΓΟ ::: 85 90 95 · »· *» · «ASP Pro Asn Tyr Leu Ala Asp Leu lie Ala Thr val Asn Ala lie Thr:: loo 105 no

Gin Lys Gly Ala Tyr Ala val Asp Pro His Asn Tyr Gly Arg Tyr ::: ii5 120 125 ··· · »· · *** Tyr Asn Ser lie lie ser Ser Asp Phe Gin Thr Phe Trp Lys. :. 130 135 140 ···· «··

Thr val Ala Ser Gin Phe Ala Ser Asn pro Leu val lie Phe Asp Thr • 145 150 155 160 ·· · · * * ♦ * ·

Asn Asn Glu Tyr His Asp Met Asp Gin Thr Leu val Leu Asn Leu Asn page 2 119434 sekluetxt.txt 165 170 175

Gin Ala Ala Ile Asp Gly Ile Arg Ser Ala Gly Ala Thr ser Gin Tyr 180 185 190

Ile Phe or Glu Gly Asn Ser Trp Thr Gly Ala Trp Thr Trp Thr Asn 195 200 205 or Asn Asp Asn Met Lys Ser Leu Thr Asp Pro Ser Asp Lys Ile 210 215 220

Tyr Glu Met His Gin Tyr Leu Asp ser Asp Gly Ser Gly Thr Ser Ala 225 230 235 240

Thr Cys Vai ser Ser Thr Ile Gly Gin Glu Arg Ile Thr Ser Ala Thr 245 250 255

Gin Trp Leu Arg Ala Asn Gly Lys Lys Gly Ile Ile Gly Glu Phe Ala 260 265 270

Gly Gly Ala Asn Asp or Cys Glu Thr Ala Ile Thr Gly Met Leu Asp 275 280 285

Tyr Met Ala Gin Asn Thr Asp or Trp Thr Gly Ala Ile Trp Trp Ala 290 295 300

Ala Gly Pro Trp Trp Gly Asp Tyr Ile Phe Ser Met Glu Pro Asp Asn 305 310 315 320

Gly Ile Ala Tyr Gin Gin Ile Leu Pro Ile Leu Thr Pro Tyr Leu 325 330 335 • · · <2io> 3

: ·: <211> 1621 *. <212> DNA

• '·· <213> Thermoascus aurantiacus' ί I <221> gene ::: <222> cd..ci62d • φ ** "* <220> <221> intron. <222> (107) .. (161) ) • · · <22o>!: <221> Intron T <222> (221) .. (280) <220>. * ··. <221> Intron * ... · <222> (434) ... (492) <220> * *. <221> intron ·: · *: <222> (558) .. (631)

Page 3 <220> <221> Intron <222> (730) .. (790) 119434 sekluetxt.txt <220> <221> Intron <222> (1549) .. (1612) <400> 3 atgaagctcg gctctctcgt gctcgctctc agcgcagcta ggcttacact gtcggcccct 60 ctcgcagaca ggaagcagga gaccaagcgt gcgaaagtat tccaatgttc gtaacatcca 120 cgtctggctt gctggcttac tggcaactga caatggcgaa gggttcggtt caaacgagtc 180 cggtgctgaa ttcggaagcc agaaccttcc aggagtcgag gtcagcatgc ctgtactctc 240 tgcattatat taatatctca agaggcttac tctttcgcag ggaaaggatt atatatggcc 300 tgatcccaac accattgaca cattgatcag caaggggatg aacatctttc gtgtcccctt 360 tatgatggag agattggttc ccaactcaat gaccggctct ccggatccga actacctggc 420 agatctcata gcggtacatt tcaattccac catgtttgga gctgtcttcg ttgtgctgac 480 atttaatggt agactgtaaa tgcaatcacc cagaaaggtg cctacgccgt cgtcgatcct 540 cataactacg gcagatagtg aggtccccgg ttctggtatt gctgctgtat atctaagtag 600 atatgtgttt ctaacatttc cacgatttca gctacaattc tataatctcg agcccttccg 660 atttccagac cttctggaaa acggtcgcct cacagtttgc ttcgaatcca ctggtcatct 720 tcgacactag taagctgaac acccgaaatt aactgagtct gag catgtct gacaagacga 780 tccatgaaag ataacgaata ccacgatatg gaccagacct tagtcctcaa tctcaaccag 840 gccgctatcg acggcatccg ttccgccgga gccacttccc agtacatctt tgtcgagggc 900 aattcgtgga ccggggcatg gacctggacg aacgtgaacg ataacatgaa aagcctgacc 960 ... *. * gacccatctg acaagatcat atacgagatg caccagtacc tggactctga cggatccggg 1020 • · · * i * acatcagcga cctgcgtatc ttcgaccatc ggtcaagagc gaatcaccag cgcaacgcaa 1080 • · tggctcaggg ccaacgggaa gaagggcatc atcggcgagt ttgcgggcgg agccaacgac 1140 * ·:.:: gtctgcgaga cggccatcac gggcatgctg gactacatgg cccagaacac ggacgtctgg 1200 actggcgcca tctggtgggc ggccgggccg tggtggggag actacatatt ctccatggag 1260 • · · ccggacaatg gcatcgcgta tcagcagata cttcctattt tgactccgta cgtacctggc 1320 cttgacggca gcaaccccgg caacccgacc accaccgtcg ttcctcccgc ttctacctcc 1380 ♦ acctcccgtc cgaccagcag cactagctct cccgtttcga ccccgactgg ccagcccggc 1440 ··· * ... · ggctgcacca cccagaagtg gggccagtgc ggcggtatcg gctacaccgg ctgcactaac 1500 m *; * tgcgttgctg gcaccacctg a cccagc t atettttctct 1560 * · «·: ***: tcccccttct agactcgctt ggatttgaca gttgctaaca tctgctcaac agtgcctgta 1620 ··· ··· ä 1621 • · · • · * * <210> 4

Page 4 119434 sekluetxt.txt <211> 415

<212> PRT

<213> Thermoascus aurantiacus <400> 4

Met Lys Leu Gly Ser Leu val Leu Ala Leu Ser Ala Ala Arg Leu Thr 15 10 15

Leu ser Ala Pro Leu Ala Asp Arg Lys Gin Glu Thr Lys Arg Lla Lys 20 25 30

Val Phe Gin Trp Phe Gly ser Asn Glu ser Gly Ala Glu Phe Gly Ser 35 40 45

Gin Asn Leu Pro Gly or Glu Gly Lys Asp Tyr He Trp Pro Asp Pro 50 55 60

Asn Thr lie Asp Thr Leu lie ser Lys Gly Met Asn He Phe Arg h 65 70 75 80

Pro Phe Met Met Glu Arg Leu val Pro Asn Ser Met Thr Gly Ser Pro 85 90 95

Asp Pro Asn Tyr Leu Ala Asp Leu He Ala Thr val Asn Ala lie Thr 100 105 110

Gin Lys Gly Ala Tyr Ala val Asp Pro His Asn Tyr Gly Arg Tyr 115 120 125

Tyr Asn Ser He He Ser Ser Pro Ser Asp Phe Gin Thr Phe Trp Lys 130 135 140 • * · * Thr Val Ala Ser Gin Phe Ala Ser Asn Pro Leu val He Phe Asp Thr ::. 145 150 155 160 • · · ♦ · • ·! * ·· Asn Asn Glu Tyr His Asp Met Asp Gin Thr Leu val Leu Asn Leu Asn. . 165 170 175 • · · · · ··· ·: 0 Gin Ala Ala lie Asp Gly He Arg Ser Ala Gly Ala Thr ser Gin Tyr ::: iso ies 190 • · «· • I * lie Phe val Glu Gly Asn Ser Trp Thr Gly Ala Trp Thr Trp Thr Asn 195 200 205 • · * • · * • · *: "*! Val Asn Asp Asn Met Lys Ser Leu Thr Asp Pro Ser Asp Lys He He 210 215 220 • · t ·· «·. · * ·. Tyr Glu Met His Gin Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Ala 225 230 235 240 9 • · * tt»: *. Thr cys val ser Ser Thr lie Gly Gin Glu Arg lie Thr Ser Ala Thr ·: ··: 245 250 255

Page 5 1 1 Q 4 λ 4 sek1uetxt.txt ^ o

Gin Trp Leu Arg Ala Asn Gly Lys Lys Gly Ile Ile Gly Glu Phe Ala 260 265 270

Gly Gly Ala Asn Asp Vai Cys Glu Thr Ala Ile Thr Gly Met Leu Asp 275 280 285

Tyr Met Ala Gin Asn Thr Asp or Trp Thr Gly Ala Ile Trp Trp Ala 290 295 300

Ala Glv Pro Trp Trp Gly Asp Tyr Ile Phe Ser Met Glu Pro Asp Asn 305 310 315 320

Gly lie Ala Tyr Gin Gin Ile Leu Pro Ile Leu Thr pro Tyr or Pro 325 330 335

Gly Leu Asp Gly Ser Asn Pro Gly Asn Pro Thr Thr Thr or Pro 340 345 350

Pro Ala Ser Thr Ser Thr Ser Arg Pro Thr Ser Ser Thr ser Ser Pro 355 360 365

Vai ser Thr Pro Thr Gly Gin Pro Gly Gly Cys Thr Thr Gin Lys Trp 370 375 380

Gly Gin Cys Gly Gly Ile Gly Tyr Thr Gly Cys Thr Asn Cys or Ala 385 390 395 400

Gly Thr Thr cys Thr Gin Leu Asn Pro Trp Tyr ser Gin cys Leu 405 410 415 ... Ί * <210> 5

: ·. <211> 1211:. · * <212> DNA

•. <213> Acremonium thermophilum: • · <220>. <221> gene <222> ¢ 13) .. C1206) C: <220> <221> intron <222> (96) .. (154) <220> ..I <221> intron <222> (404) .. (526) <220> <221> intron <222> (1134) .. (1197) • ♦ <400> 5: *; ggactgcgca tcatgcgctc ctcacccttt ctccgcgcag ctctggctgc cgctctgcct 60 *: **: ctgagcgccc recoverccctcga cggaaagtcg acgaggtatg ccaatcctcg tacctctgcc 120

Page 6 119434 sekluetxt.txt ctctgtagaa acaagtgacc gactgcaaag acagatactg ggactgctgc aagccgtcct 180 gcggctgggc cggaaaggcc tcggtgaacc agcccgtctt ctcgtgctcg gccgactggc 240 agcgcatcag cgacttcaac gcgaagtcgg gctgcgacgg aggctccgcc tactcgtgcg 300 ccgaccagac gccctgggcg gtcaacgaca acttctcgta cggcttcgca gccacggcca 360 tcgccggcgg ctccgagtcc agctggtgct gcgcctgcta tgcgtgagtt ctctgcaagc 420 cgcttcccac ccccgctttc tgtgcaggcc gcttcccccc tacccaccca cttccccccc 480 cccgcctctg tgatcgggca tccgagctaa gttgcgtgtc gtccagactc accttcaact 540 cgggccccgt cgcgggcaag accatggtgg tgcagtcgac cagcaccggc ggcgacctgg 600 gcagcaacca gttcgacctc gccatccccg gcggcggcgt gggcatcttc aacggctgcg 660 cctcccagtt cggcggcctc cccggcgccc agtacggcgg catcagcgac cgcagccagt 720 gctcgtcctt ccccgcgccg ctccagccgg gctgccagtg gcgcttcgac tggttccaga 780 acgccgacaa ccccaccttc accttccagc gcgtgcagtg cccgtccgag ctcacgtccc 840 gcacgggctg taagcgcgac gacgacgcca gctatcccgt cttcaacccg cctagcgtcc 900 ctggccttga cggcagcaac cccggcaacc cgaccaccac cgtcgttcct cccgctt cta 960 cctccacctc ccgtccgacc agcagcacta gctctcccgt ttcgaccccg actggccagc 1020 ccggcggctg caccacccag aagtggggcc agtgcggcgg tatcggctac accggctgca 1080 ctaactgcgt tgctggcacc acctgcactc agctcaaccc ctggtacagc caggtatgtt 1140 tctcttcccc cttctagact cgcttggatt tgacagttgc taacatctgc tcaacagtgc 1200 ctgtaactgc a 1211 <210> 6. <211> 315

• J · <212> PRT

"** <213> Acremonium thermophilum • · · *. * <400> 6 • I *. * / Met Arg ser ser Pro Phe Leu Arg Ala Ala Leu Ala Ala Ala Leu Pro ::: 1 5 10 15 ··· «• · ·" · Leu Ser Ala His Ala Leu Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys :: 20 25 30 ···. Cys Lys Pro Ser Cys Gly Trp Ala Gly Lys Ala Ser Val Asn Gin Pro::: 35 40 45 ·· «♦ ·· • ·” · * Val Phe Ser cys Ser Ala Asp Trp Gin Arg Ile Ser Asp Phe Asn Ala. :. 50 55 60 »4 **

• M

Lys ser Gly Cys Asp Gly Gly Ser Ala Tyr ser Cys Ala Asp Gin Thr 65 70 75 80 • * · I t • · ....: pro Trp Ala val Asn Asp Asn Phe ser Tyr Gly Phe Ala Thr Ala page 7 119434 sekluetxt.txt ° ^ 85 90 95

Ile Ala Gly Gly Ser Glu Ser ser Trp cys cys Ala Cys Tyr Ala Leu

100 105 HO

Thr Phe Asn ser Gly Pro or Ala Gly Lys Thr Met or Vai Gin ser 115 120 125

Thr ser Thr Gly Gly Asp Leu Gly ser Asn Gin Phe Asp Leu Ala Ile 130 135 140

Pro Gly Gly Gly or Gly Ile Phe Asn Gly cys Ala Ser Gin Phe Gly 145 150 155 160

Gly Leu Pro Gly Alla Gin Tyr Gly Gly Ile Ser Asp Arg Ser Gin Cys 165 170 175

Ser ser Phe Pro Ala Pro Leu Gin Pro Gly Cys Gin Trp Arg Phe Asp 180 185 190

Trp Phe Gin Asn Lower Asp Asn Pro Thr Phe Thr Phe Gin Arg Vai Gin 195 200 205

Cys Pro Ser Glu Leu Thr ser Arg Thr Gly Cys Lys Arg Asp Asp Asp 210 215 220

Ala ser Tyr pro or Phe Asn pro Pro ser or Pro Gly Leu Asp Gly 225 230 235 240

Ser Asn Pro Gly Asn Pro Thr Thr Thr or Vai Pro Pro Ala Ser Thr 245 250 255 • * * "'l Ser Thr Ser Arg Pro Thr Ser Ser Thr Ser Ser Pro or Ser Thr pro: * ··; 260 265 270« · • · • · ♦ * · Thr Gly Gin Pro Gly Gly Cys Thr Thr Gin Lys Trp Gly Gin Cys Gly: 275 280 285 1 · «· 0 • · ·

Gly Ile Gly Tyr Thr Gly Cys Thr Asn cys or Ala Gly Thr Thr cys · * '*: 290 295 300 ···

Thr Gin Leu Asn Pro Trp Tyr ser Gin Cys Leu: 305 310 315 »• f *» · • · '·· * <210> 7 • <211> 1663

<212> DNA

<213> Chaetonrium thermophilum • · • · · <220> I <221> gene <222> Cl) .. (1663)

Page 8 <220> <221> Intron <222> (1591) .. (1654) sekluetxt.txt 119 434 <400> 7 atgatgtata agaagttcgc cgctctcgcc gccctcgtgg ctggcgcctc cgcccagcag 60 gcttgctccc tcaccgctga gaaccaccct agcctcacct ggaagcgctg cacctctggc 120 ggcagctgct cgaccgtgaa cggcgccgtc accatcgatg ccaactggcg ctggactcac 180 accgtctccg gctcgaccaa ctgctacacc ggcaaccagt gggatacctc cctctgcact 240 gatggcaaga gctgcgccca gacctgctgc gtcgatggcg ctgactactc ttcgacctat 300 ggtatcacca ccagcggtga ctccctgaac ctcaagttcg tcaccaagca ccagtacggc 360 accaacgtcg gctcccgtgt ctatctgatg gagaacgaca ccaagtacca gatgttcgag 420 ctcctcggca acgagttcac cttcgatgtc gatgtctcca acctgggctg cggtctcaac 480 ggcgccctct acttcgtttc catggatgct gatggtggca tgagcaaata ctctggcaac 540 aaggctggcg ccaagtacgg taccggctac tgcgatgctc agtgcccgcg cgacctcaag 600 ttcatcaacg gcgaggccaa cgttgggaac tggaccccct cgaccaacga tgccaacgcc 660 ggcttcggcc gctatggcag ctgctgctct gagatggatg tctgggaggc caacaacatg 720 gctactgcct tcactcctca cccttgcacc accgttggcc agagcgg ctacag ctctgaccgc tatgctggtg tttgcgaccc tgatggctgc 840 gacttcaacg cctaccgcca aggcgacaag accttctacg gcaagggcat gactgtcgac 900 accaacaaga agatgaccgt cgtcacccag ttccacaaga actcggctgg cgtcctcagc 960 gagatcaagc gcttctacgt ccaggacggc aagatcattg ccaacgctga gtccaagatc 1020 cccggcaacc ccggaaactc cattacccag gagtattgcg atgcccagaa ggtcgccttc 1080.:. agtaacaccg atmacttcaa ccgcaagggc ggtatggctc agatgagcaa ggccctcgca 1140 »··». * ·. *. ggccccatgg tcctggtcat gtccgtctgg gatgaccact acgccaacat gctctggctc 1200 • · *. ·. : gactcgacct accccatcga ccaggccggc gcccccggcg ccgagcgcgg tgcttgcccg 1260 • * ·:. ·, accacctccg gtgtccctgc cgagatcgag gcccaggtcc ccaacagcaa cgtcatcttc. . ·. tccaacatcc gtttcggccc catcggctcg accgtccctg gccttgacgg cagcaacccc 1380 «··. * · *. ggcaacccga ccaccaccgt cgttcctccc gcttctacct ccacctcccg tccgaccagc 1440 * * • * · agcactagct ctcccgtttc gaccccgact ggccagcccg gcggctgcac cacccagaag 1500. . ·. tggggccagt gcggcggtat cggctacacc ggctgcacta actgcgttgc tggcaccacc 1560 ··· *. * ··. tgcactcagc tcaacccctg gtacagccag gtatgtttct cttccccctt ctagactcgc 1620 · »· ttggatttga cagttgctaa catctgctca acagtgcctg taa 1663 • · φ ·· * · <2io> 8 *: * <211> 1076

• V. <212> DNA

:, · <213> Acremonium thermophilum

Page 9 <220> <221> gene <222> Cl) (1076) 119434

sekluetxt.txt 1 1 7 H J H

<220> <221> Intron <222> (84) .. (142) <220> <221> Intron <222> (392) .. (514) <400> 8 recovercgctcct caccctttct ccgcgcagct ctggctgccg ctctgcctct gagcgcctggggcagcg aatcctcgta cctctgccct ctgtagaaac 120 aagtgaccga ctgcaaagac agatactggg actgctgcaa gccgtcctgc ggctggccgg 180 gaaaggcctc ggtgaaccag cccgtcttct cgtgctcggc cgactggcag cgcatcagcg 240 acttcaacgc gaagtcgggc tgcgacggag gctccgccta ctcgtgcgcc gaccagacgc 300 cctgggcggt caacgacaac ttctcgtacg gcttcgcagc cacggccatc gccggcggct 360 ccgagtccag ctggtgctgc gcctgctatg cgtgagttct ctgcaagccg cttcccaccc 420 ccgctttctg tgcaggccgc ttccccccta cccacccact tccccccccc cgcctctgtg 480 atcgggcatc cgagctaagt tgcgtgtcgt ccagactcac cttcaactcg ggccccgtcg 540 cgggcaagac catggtggtg cagtcgacca gcaccggcgg cgacctgggc agcaaccagt 600 tcgacctcgc catccccggc ggcggcgtgg gcatcttcaa cggctgcgcc tcccagttcg 660 gcggcctccc cggcgcccag tacggcggca tcagcgaccg cagccagtgc tcgtccttcc 720 ccgcgccgct ccagccgggc tgccagtggc gcttcgactg gttccagaac gccgacaacc 780 ccaccttc ac cttccagcgc gtgcagtgcc cgtccgagct cacgtcccgc acgggctgta 840 ·· * agcgcgacga cgacgccagc tatcccgtct tcaacccgcc tagcggtggc tcccccagca 900 • * · · * ccaccagcac caccaccagc tccccgtccg gtcccacggg caaccctcct ggaggcggtg 960 • * ·] · gctgcactgc ccagaagtgg gcccagtgcg gcggcactgg cttcacgggc tgcaccacct 1020

t »I

··· * gcgtctcggg caccacctgc caggtgcaga accagtggta ttcccagtgt ctgtga 1076 • · «• * ♦ ··. ** ·. <210> 9 * · ** <211> 45

<212> DNA

<213> Thermoascus aurantiacus <400> 9 · * '*. attaaccgcg gactgcgcat catgaagctc ggctctctcg tgctc 45 «· *: * <2io> ίο * ::: <2n> 29

<212> DNA

"* <213> Thermoascus aurantiacus ·· ·: V <400> 10 ....: aactgaggca tagaaactga cgtcatatt 29 • ·

Page 10 sekluetxt.txt ^ 79434

<210> 11 <211> 28 <212> DNA

<213> Chaetomium thermophilum <400> 11 taatttacgt acctggcctt gacggcag 28 <210> 12 <211> 30

<212> DNA

<213> Chaetomium thermophilum <400> 12 attaactgca gttacaggca ctgttgagca 30 <210> 13 <211> 42

<212> DNA

<213> Acremonium thermophilum <400> 13 attaaccgcg gactgcgcat catgcgctcc tcaccctttc tc 42 <210> 14 <211> 33

<212> DNA

<213> Acremonium thermophilum <400> 14 ttaatctgca gtcacagaca ctgggaatac cac 33 <210> 15

<211> 81 <212> PRT

<213> Chaetomium thermophilum ... t <400> 15 • · · • * · * val Pro Gly Leu Asp Gly ser Asn pro Gly Asn pro Thr Thr Thr val. 1 5 10 15 • · · • ·· • • • •. · I val Pro Pro Ala Ser Thr Ser Thr ser Arg pro Thr ser Ser Thr Ser 20 25 30 • * «* · · ··· • •! Ser Pro Val Ser Thr Pro Thr Gly Gin Pro Gly Gly Cys Thr Thr Gin 35 40 45 I:: Lys Trp Gly Gin cys Gly Gly He Gly Tyr Thr Gly cys Thr Asn cys ... 50 55 60 • · • · «• 9 .:. val Ala Gly Thr Thr cys Thr Gin Leu Asn Pro Trp Tyr Ser Gin cys -: 65 70 75 80 ··· • · • 9 • · · • Leu 99 9 • · 9 • 9 • · «•» * «• ·

Page 11

Claims (23)

119434 36 A cellulase fusion protein, characterized in that it comprises an amino acid sequence of a first endoglucanase nucleus having at least 5 75% identity to SEQ ID NO: 2 or a fragment thereof having cellulase activity and a second amino acid sequence comprising a linker and a cellulose binding domain (CBD). having at least 75% identity to SEQ ID NO: 15 or a fragment thereof having cellulose binding activity, Fusion protein according to claim 1, characterized in that the endoglucanase nucleus is derived from Thermoascus aurantiacus, in particular T, aurantiacus CBS 116239. Fusion protein according to claim 1, characterized in that it comprises amino acids 19-334 of SEQ ID NO: 2. Fusion protein according to claim 1, characterized in that the linker and the CBD are derived from the cellobiohydrolase of Chaetomium thermophilum, in particular C. thermophilum CBS 730.95. The fusion protein according to claim 1, characterized in that the endoglucanase core comprises the sequence of SEQ ID NO: 2 and the linker and CBD comprise the sequence of SEQ ID NO: 15. The fusion protein of claim 1, characterized in that it comprises the sequence of SEQ ID NO: 4 or a fragment thereof: having cellulase activity and cellulose binding activity. 7. The fusion protein of claim 1, characterized in that the endoglucanase core is encoded by a gene corresponding to E. coli DSM ···. ··. 17326. A fusion protein according to claim 1, characterized by . that it is encoded by a fusion gene corresponding to the gene contained in E. coli DSM 18159. '*: ** 30 An isolated polynucleotide, characterized in that it is selected from the group consisting of: (a) the nucleotide sequence of SEQ ID NO: 3 or the fusion protein coding sequence of claim 1 (b) (a); ) complementary waistband; (C) a sequence degenerate with respect to the genetic code from any of the sequences defined in (a) or (b). 119434 37 A polynucleotide according to claim 9, characterized in that it comprises a fusion gene corresponding to a gene included in E. coli DSM 18159. Expression vector, characterized in that it comprises the nucleotide sequence according to claim 9 of claim 5. A host cell, characterized in that it comprises an expression vector according to claim 11. Host cell according to claim 12, characterized in that it is a fungus, in particular of the genus Trichoderma. A method for producing a fusion protein according to any one of claims 1 to 8, characterized in that the method comprises transforming the host cell with an Expression vector encoding the fusion protein and culturing the host cell under conditions allowing expression of the fusion protein and optionally recovering and purifying it. An enzyme preparation, characterized in that it comprises a fusion protein according to any one of claims 1 to 8. A process for treating cellulosic material, characterized in that the process comprises contacting the cellulosic material with a fusion protein according to any one of claims 1 to 8 or with an enzyme preparation according to claim 15. • · · ;; * j 17. A process according to claim 16, characterized in that: the cellulosic material is a textile material for use in animal feed: plants or pulp of wood or recycled fiber. :. · · 25 A process according to claim 16, characterized in that: extracting the vegetable oil. A biofuel washing process, characterized in that the process comprises contacting the denim fabric or garments with a fusion protein according to any one of claims 1 to 8 or an enzyme preparation according to claim 11. The biofinishing process, wherein the process comprises the steps of contacting a textile material such as fabrics, garments or yarns with the fusion protein, ···, n or any of claims 15 to 15. with the enzyme preparation. · 119434 38 Detergent, characterized in that it comprises a fusion protein according to any one of claims 1 to 8 or an enzyme preparation according to claim 15 and detergent adjuvants. Animal feed, characterized in that it comprises the fusion protein according to any one of claims 1 to 8. 23. Escherichia coli strain, characterized by the accession number DSM 18159. I *. *. * · · · · · · · · · · · · · · · · · · · · · · · · · · ··· · «• · · · · · · ··· I · • * f · • · 119434 39