FI119325B - New endoglucanase polypeptides and their preparation and use - Google Patents

Description

119325 ί

Novel endoglucanase polypeptides and their production and use Field of the Invention

The present invention relates to novel cellulase enzymes, in particular novel endoglucanases, preparations and compositions containing these endoglucanase enzymes, expression vectors, host cells and methods for their preparation, and to the use of cellulases, preparations and compositions in the textile, detergent and pulp and paper industries.

Background of the Invention

Cellulose is a major structural component of higher plants and naturally occurs in almost pure form only in cotton fibers. It provides plant cells with high tensile strength, helping them to resist mechanical loading and osmotic pressure. Cellulose is a linear polysaccharide of glucose linked by β-1,4 bonds. In nature, cellulose is generally incorporated into lignin together with hemicelluloses such as xylans and Glu-15 commandants. Cellulolytic enzymes hydrolyze cellulose and are produced by a wide variety of bacteria and fungi. Cellulases are industrially important enzymes with a current annual market value of approximately US $ 190 million. In the textile industry, cellulases are used in finishing denim to create a fashionable stone-washed appearance on denim fabrics. ·, · 20 in the bi-stone washing method and are also used, for example, to remove linen and to prevent bumps on cotton garments. Cellulases are used in the pE **. * Diluent industry to brighten colors and prevent graying and flaking of clothing. Cellulases are still used: ··: in the food and animal feeds industry and · ·: 25 have great potential in the pulp and paper industry, for example in tamping to decolourize fiber surfaces and improve dewatering wear. The wide spectrum of industrial uses for cellulase has formed: Y need for commercial cellulase products containing various types of cellulose • ·. ***. glass components and work optimally at different pH and temperature ranges.

The practical use of cellulases is hampered by the nature of the known cellulase * * * * · [· * glass compositions, which are often enzyme mixtures with multiple activities and substrate specificities. For this reason, efforts have been made to: * <*: land cellulases with only the desired activities. The unique properties of each cellulase make some cellulases more suitable for certain purposes than others. When enzymes differ in many ways, one of the 2 119325 major differences is pH optimum. Neutral cellulases are most active at pH 6-8 and alkaline cellulases at pH 7.5-10, while acidic cellulases with a pH optimum of 4.5-5.5 show very low levels of activity at higher pH- values. Neutral and acidic cellulases 5 are particularly useful in the textile industry. In fabric treatment, cellulases attack cotton fiber-forming chains of cellulose molecules in this way, affecting fabric characteristics.

In the textile industry, the "stone-washed" appearance or worn-out appearance has been of interest to denim producers in recent years. worn out ”appearance. Controlled enzyme treatments result in less damage to clothing and machinery and eliminate the need for stone destruction.

In addition, the textile industry uses cellulases in biofinishing, i.e. create a permanent improvement in anti-peeling and improved durability against peeling, a reduced pile clarified texture, a better feel of textiles such as softness, smoothness and silky feel 20, improved descent, and brighter colors and improved moisture absorption.

The cellulases used for the treatment of denim are generally divided into two main groups: * acidic and neutral cellulases. Acid cellulases. typically operate at pH 4.5 to 5.5 and neutral cellulases at pH 6-8.

: i *. 25 Acidic cellulases used in biofuel washing are mainly derived from Trichoder • · J'V ma reesees (genus Hypocrea jecorina) and neutral cellulases from various fungi including Melanocarpus, Humicola, Myce-, Fusarium, Fusarium, Acremonium and Chrysosporium (Haakana et al. 2004).

T. reesei enzymes include, for example, cellulases from the glycoside family 5 (endo-:? 30 glucanase II, EGII), family 7 (cellobiohydrolase I, CBHI) and family 12 ··· (endoglucanase III, EGIII; Ward et al. 1993) and neutral cellulases, most-: v. min. endoglucanases, from family 45 and family 7 (Henrissat, 1991; Henry., ···. sat and Bairoch, 1993).

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Cellulases comprise a catalytic domain / core (CD) that expresses cellulase activity. In addition to the catalytic domain, the cellulase molecule may comprise one or more cellulose binding domains (CBDs), also referred to as carbohydrate binding domains / modules (CBD / CBM), which may be located at either the N or C terminus of the catalytic domain. CBDs have carbohydrate binding activity and mediate cellulase binding to crystalline cellulose but have little or no effect on the hydrolytic activity of the cellulase enzyme against soluble substrates. These two domains typically combine via a flexible and highly Gly cosylated linker.

Cellulases, which mainly attack the surface of the fiber, are particularly useful for stone washing of denim-dyed denim because color 10 is located on the surface of the fiber. When used to treat cotton fabrics, acidic cellulases generally require a shorter wash time than neutral cellulases. Acidic cellulases are used particularly in bio-finishing (anti-peeling) and also in denim treatment (bio-stone washing).

In the context of the present invention, endoglucanases (EGs) refer to E.C. 3.2.1.4 are enzymes classified as one of the three cellulase types commonly required for the biological conversion of cellulose to glucose. Endoglucanases cleave internal beta-1,4-glucosidic bonds, while cellobiohydrolases cleave disaccharide cellobiose at the end of the cellulose polymer chain, and beta-1,4-glucosidases hydrolyze cellobiose and other short cello-oligosaccharides to glucose. Some naturally occurring endoglucanases have a cellulose binding domain (CBD) while others do not.

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Endoglucanases are also widely used in textiles, detergents, and. in the pulp and paper industry. For example, the endoglucose of the cel45 family: ·. 25 nases (EG family 45) are described, for example, in U.S. Patent No. 6,001,639, which discloses enzymes having endoglucanase activity and two conserved amino acid sequences. Uses in textile, detergent and fiber pulp and paper applications are widely discussed and the treatment of lignocellulosic material is mentioned. WO 2004/053 039 is directed to end-glucanase pe-30 agent applications. U.S. Patent No. 5,958,082 discloses the use of endoglucanase, particularly from Thiela &lt; / RTI &gt; terrestris, in textile applications to provide a stone-washed or scratched appearance of twilight jeans. EP 0 495 258 relates. detergent compositions containing Humicola cellulase. U.S. Patent No. 5,948,672 describes a cellulase preparation containing endoglucanase, especially from Humicola, and its use in textile and fiber pulp applications.

t · 4 119325 EGs and EG enriched compositions and concentrates are also commercially available.

However, there is a continuing need for improved cellulases, including endoglucanases, which are more effective in fabric treatment and other areas where cellulases are traditionally used. In particular, there is a continuing need for more efficient cellulases to improve the economics of the process.

The present invention aims to meet this need.

Brief Description of the Invention

It is an object of the present invention to provide novel endoglycan channels having improved hydrolytic properties for use in the textile industry, particularly in cotton finishing, such as anti-soiling and stonewashed denim, and for use in detergent compositions and other fields. The novel endoglucanases of the invention have the advantage of being active at acidic and neutral pHs, having a greatly improved potency in Textile bio-finishing and bio-stone washing applications and detergent applications. When used in the treatment of cellulose-containing textile materials, the new endoglucanases provide a smooth feel, improved appearance and softness, and a permanent anti-peeling effect on textiles. Due to the improved efficiency of the endoglucanases of the invention, the use of enzymes is significantly more economical. Additional advantages are also achieved in the logistics and storage of enzyme products when smaller quantities of the enzyme product are required. In addition, the endoglucanases of the present invention, when acidic, act faster to produce times

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; '' A cost-effective handling procedures and savings in equipment and conceptual: * V 25 hardware.

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

It is a further object of the present invention to provide novel expression plasmids or vectors containing polynucleotides useful for the production of novel endoglu-· · 'M' canases of the present invention and novel * * transformed with these expression plasmids. · * Hosts.

It is a further object of the present invention to provide ent- *: **: 35 enzyme preparations containing one or more endoglucanases having improved hydrolytic properties.

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It is a further object of the present invention to provide methods of using enzyme preparations and endoglucanases for the finishing of textiles, in particular denim bio-finishing and bio-stone washing.

It is a further object of the present invention to provide means 5 for using the enzyme preparations of the invention in detergent compositions.

The present invention relates to an endoglucanase polypeptide comprising a fragment having cellulolytic activity and selected from the group consisting of: a) a polypeptide comprising at least 80% identity to SEQ ID NO: 2; and b) a fragment of a) having cellulolytic activity.

The present invention further relates to an isolated polynucleotide encoding an endoglucanase polypeptide as defined above selected from the group consisting of: a) a nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO: 18; (b) the complementary strand of (a); c) a fragment of at least 20 nucleotides according to a) or b); and d) a sequence degenerate with respect to the genetic code of any one of the sequences defined in a), b) or c).

The present invention further relates to an expression vector comprising a polynucleotide sequence as defined above.

•. *. The present invention still further relates to novel # · Ι Ι:. Hosts transformed with vectors, in particular hosts capable of high expression levels of the endoglucanase of the invention.

The present invention further relates to an enzyme preparation containing one or more endoglucanases of the invention.

The present invention further relates to methods of using the enzyme preparations according to the invention for the biological finishing of textiles, in particular for the inhibition of nymphs.

m. *. *. The present invention further relates to the enzyme ·· * of the invention. • methods of using finished products for finishing textiles, in particular denim • · stonewashing.

The present invention further relates to the use of the enzyme preparations of the invention in detergent compositions.

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Brief Description of the Drawings

Figure 1 illustrates a schematic representation of the expression cassettes used to transform Trichoderma reesei protoplasts for the production of the Acramonium thermophilumm cellulases of the invention. The recombinant genes were under the control of the T. reesei cbhl / cel7A promoter (cbhl prom) and transcription termination was confirmed by the addition of the T. reesei dbh1 terminator (cbhl term). The amdS gene (amdS) was included for the selection of transformants.

Figure 2 illustrates the pH dependence of heterologously produced A. thermophilum EG_40 cellulase by assaying the culture supernatant using CMC as a substrate for a 10 minute reaction at 50 ° C (A). The temperature optimum of EG_40 cellulase was determined at optimal pH (5.5). The reaction with CMC as substrate was carried out for 60 minutes. BSA (100 µg / ml) was added as a stabilizer. (B).

Figure 3 shows a comparison of the bioavailability / abrasion level of the novel endoglucanase of the invention with the EGII-enriched concentrate of the prior art. The bio-stone wash effect was evaluated by measuring the color.

Figure 4 shows a comparison of the anti-Nyp decay properties of the novel endoglucanase of the invention with the prior art EGII-enriched concentrate.

Figure 5 shows a series of photographs showing the anti-pitting effect of the endoglucanase of the invention.

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DETAILED DESCRIPTION OF THE INVENTION • · ·. . ·. The present invention is based on efforts to further improve:. cellulases for use in the textile industry. Surprisingly, it was found that, starting with Acremonium, new endoglucanases could be isolated and recombinant enzymes could be produced that not only have an annoying temperature profile but also exhibit surprisingly favorable napping. and at least four times as effective as: .v commercial EG containing formulation. In addition, the new endoglucanases showed * · ♦ 30 excellent bio-stone washing properties compared to prior art. *. *. cellulases.

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Accordingly, the present invention relates to an endoglucanase poly-peptide comprising a fragment having cellolytic activity selected from the group consisting of? 7119325 a) a polypeptide comprising at least 80% amino acid sequence; identity with SEQ ID NO: 2; b) a fragment of a) having cellulolytic activity.

In one preferred embodiment of the invention, said amino acid has at least preferably 85%, more preferably 90%, even more preferably 95% and most preferably 98% identity to SEQ ID NO: 2.

In a preferred embodiment of the invention, said endoglucanase has SEQ ID NO: 2.

In yet another preferred embodiment of the invention, said endoglucanase has the amino acid sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

In yet another preferred embodiment of the invention, the polypeptides are obtainable from or derived from Acremonium sp., Preferably Acremonium thermophilum.

Also described herein is an endoglucanase fusion protein comprising an amino acid sequence derived from a polypeptide comprising an amino acid sequence having at least 68% identity to SEQ ID NO: 4 linked to a cellulose binding domain (CBD). Preferably it has at least 70%, preferably 75%, more preferably 80%, even more preferably 85%, still more preferably 90% and most preferably 95% identity to SEQ ID NO: 4. Preferably, said fusion protein further comprises a fusion region. Preferably, said fusion protein comprises the amino acid sequence of SEQ ID NO: 4 linked to a CBD derived from a polypeptide comprising an amino acid. a further sequence of SEQ ID NO: 2. More preferably, said endoglucanase fusion protein has SEQ ID NO: 21.

The present invention further relates to an isolated polynucleotide encoding an endoglucanase polypeptide as defined above.

***** In one embodiment of the invention, the isolated polynucleotide has a nucleotide sequence selected from the group consisting of: v. A) a nucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO: 18; . *. *. (b) the complementary strand of (a); * ··. c) a V fragment of at least 20 nucleotides according to a) or b); and: V 35 d) a sequence degenerate with the genetic code of any of the sequences defined in a), b) or c).

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The present invention further relates to an expression vector comprising a polynucleotide sequence as defined above.

The present invention further relates to novel hosts transformed with the vectors of the invention, in particular hosts capable of high levels of expression of the endoglucanase of the invention. According to a preferred embodiment of the invention, the enzymes are available from Acremonium thermophilum strain ALK04245, which is deposited under accession number CBS 116240.

The present invention further relates to an enzyme preparation containing one or more endoglucanases of the invention.

The present invention further relates to methods of using the enzyme preparations of the invention for the biological finishing of textiles, in particular for inhibiting nyp-catching.

The present invention further relates to methods of using the enzyme preparations of the invention for finishing textiles, in particular denim 15 for stone washing.

The present invention further relates to the use of the enzyme preparations of the invention in detergent compositions.

The endoglucanase protein formulations of the invention are particularly useful in the textile and detergent industry. They are particularly useful in the textile industry for bio-finishing fabrics or garments, such as anti-peeling, anti-linting, color brightening, reducing roughness, creating various finishes (for example: *: * · 'peachy', 'worn', 'sand' or ' old appearance) and. . *. biofinishing of yarns, eg to reduce fluffiness and improve smoothness:. Additional uses include use in detergent compositions to improve the fabric's treatment properties by anti-peeling, anti-graying, color brightening and softening, and to improve the cleaning effect of textiles, for example to remove dirt. Additional uses still include use in denim bio-stone washing.

v .: 30 In cotton fabric, the lint (microfibres) emerges from the surface and ···! ...: can be wound together during processing to form bumps. Enzymes: v. weaken the microfibers that rise up from the surface and are then removed by the shear forces of the treatment (Nierstrasz and Warmoeskerken, 2003). As used in the present context, the term "biofinishing" (also called nip-: V 35 catch inhibition, antifouling, or biopolishing) refers to the use of enzymes in the controlled hydrolysis of cellulosic fibers to modify the surface of a fabric or 9 119325 yarn, which permanently prevents peeling, improves fabric feel such as softness and smoothness, clarifies surface structure by reducing lint, resulting in color clarification, improves fabric settling, improves moisture absorption, which can also improve dyeing. Cellulase enzymes are used for the treatment or finishing of cellulose-containing textile materials such as cotton, flax, jute, jute, viscose, modal fibers, lyocell and cupro, or mixtures thereof.

As used herein, the term "bio-stone washing" of a fabric or garment refers to the use of enzymes instead of pumice stones, or in addition to the treatment of the fabric or garment, especially denim.

As used herein, the term "back dyeing" refers to the tendency of the released color to re-deposit on the surface of the fabric fibers.

15 As used herein, the term "detergent" refers to a cleaning agent that may contain surfactants (anionic, nonionic, cationic, or ampholytic surfactants), fillers, and other optional ingredients such as anti-redeposition and soil suspending agents, optical brighteners. , bleaches, dyes and pigments and hydrolase-20 and. Suitable listing of detergent contents is given in U.S. Patent No. 5,433,750, and suitable listing of surfactants is given in U.S. Patent No. 3,664,961.

The biological activity of endoglucanase is its catalytic activity. · *: ·! and / or its ability to bind cellulosic material. The cellulolytic activity of endoglucanase is its hydrolytic activity.

• · «. **: 25 As used herein, the term" Acremonium ϊ * γ sp. "Refers to a filamentous genus having the characteristics of CBS 116240.

· · ·: As used in the present context, a “polynucleotide” refers to both RNA and DNA and may be single-stranded or double-stranded. The polynucleotide may also be a fragment of said polynucleotides comprising: V: 30 at least 20 nucleotides, e.g. at least 25, 30 or 40 nucleotides.

According to one embodiment of the invention, it has a length of at least 100 µm, 200 or 300 nucleotides. Further, the polynucleotide may be degenerate as a result of the genetic code into one of the sequences defined above. This means that different codons can encode the same amino acid.

: ***: 35 The novel polypeptides may also be variants of said polypeptides. A "variant" may be a polypeptide that occurs naturally, eg, as an allelic variant within the same strain, species, or genus, or may be degenerate by mutagenesis. It may comprise amino acid substitutions, deletions, or insertions, but it still functions in substantially the same way for the enzymes defined above, i.e. it comprises a fragment having cellulolytic activity.

The expression vector is a cloning plasmid or vector capable of expressing DNA encoding the endoglucanases and endoglucanase fusion proteins of the invention after transformation into the desired host. When the fungal host is used, the gene of interest is preferably transmitted to the fungal host as part of a cloning or expression vehicle that integrates with the fungal chromosome or allows the gene of interest to be integrated into the host chromosome, or independently replicated. Sequences that are part of a cloning transporter or expression transporter may also be integrated into said DNA during the integration process. In addition, the expression vector or portions thereof in the sponge may be targeted at predetermined sites.

The DNA encoding the endoglucanases of the invention and the en-doglucanase fusion proteins are also preferably placed under the control (i.e., cleaved) of certain control sequences mediated by a vector (which integrates with the gene of interest), such as promoter sequences. Alternatively, the control sequences may be those at the insertion site.

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The sequences controlling expression of the expression vector will vary depending on whether the vector is designed to express a particular gene in a prokaryotic or eukaryotic host (for example, the shuttle vector may transmit the! Y gene for selection of bacterial hosts). Expression-controlling 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 i.v. 30 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' γ * are 'operably linked' to the nucleotide sequence encoding the polypeptide: slide.

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A cleavable linkage is a linkage in which a sequence is linked to a control sequence (or sequences) in such a way that expression of the sequence is placed under the control or control of a regulatory sequence. Two DNA sequences (such as the promoter region sequence fused to the 5 'end of the 5 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 operably linked regulatory elements, such as enhancer sequences.

In a preferred embodiment, genetically stable transformants are coprostrated to integrate the DNA encoding the endoglucanases and endogenucanase fusion proteins of the invention into a host chromosome by transformation with a vector that protects sequences that promote integration of said vector into the chromosome.

Cells having DNA encoding the endoglucanases or endoglu-15 channelase fusion proteins of the invention stably integrated into their chromosome are also selected by the addition of one or more homologous or heterologous markers that allow selection of host cells containing the expression vector, e.g., markers. antibiotics of resistance or heavy metals such as copper, or mark-20 bodies that complement the auxotrophic mutation in the host chromosome, etc. The selectable marker gene can either be directly linked to the expressed DNA gene sequence or introduced into the same cell by co-transformation.

When the vector or DNA sequence of the invention containing the construct (s) is prepared for expression, the DNA constructs are introduced into the appropriate host cell by any of a variety of suitable means, including by transformation known in the art. Receiving cells for vector introduction are cultured in a selective medium that selects: ···: transformed cells for growth.

Suitable host systems for expression and production include, for example, a production system for the fungal Trichoderma system (EP 244 234) or an Aspergillus production system such as A. oryzae or A. niger (WO 9 708 325 and WO 9 533 386). No. 5,843,745, No. 5,770,418) or a production system developed for Fusarium, such as F. oxysporum (Malardier et al., 1989). Suitable production systems for bacteria are the production system developed for Bacillus, for example, B. subtiles, or E. Sol or actinomycete Streptomy - *: **. Suitable production systems for yeasts are for Saccharomyces, 12 119325

Systems developed for Shlzosaccharomyces or Pichia pastoris. Production systems in some other microbes or in mammalian cells or plants are also possible.

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

Fragments are understood to be portions of polypeptide or nucleic acid molecules long enough to have the desired enzymatic properties or to encode the desired endoglucanases or endoglucanase fusion proteins or a biologically active fragment thereof. The term &quot; derivative &quot; as used herein means that the nucleotide sequences of these molecules differ from the sequences of the above described nucleic acid molecules at one or more sites and are highly homologous to said sequences.

As used herein, the term "identity" refers to a global identity between two amino acid sequences relative to each other from the first amino acid encoded by the corresponding gene to the last amino acid. Rice et al., 2000), · * ·,: version 3.0.0, with the following parameters: EMBLOSUM62, gap penalty 10.0, intrinsic penalty 0.5 The algorithm is described by Needleman and Wunsch (1970). The results obtained using the -Wunsch ai · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 25 gormitory are only comparable when aligning the corresponding domains of the Sequence. of which: ···: these elements are missing , can't be done.

Cellulolytic enzymes useful for hydrolysis of cellulosic material are available from or derived from Acremonium sp., Preferably A.

thermophilum. “Available” or “derived from” means that they can be obtained. · * ·. From the species mentioned, but does not exclude the possibility of obtaining them from other sources. • · · M. In other words, they may be derived from any **: ** including plants Preferably they are derived from microorganisms, e.g.

j * 35 bacteria or fungi. The bacteria may be, for example, from the genus selected from *: **: Bacillus, Azospirillum and Streptomyces. More preferably, the enzyme is 13,119,325 derived from fungi (including filamentous fungi and yeasts), for example, a family selected from the group consisting of Thermoascus, Acremonium, Chaetomium, Achaetomium, Aspergillus, Botrytis, Chry-sosporium, Fly, Hypocreas-5, Lentinus, Melanocarpus, Myceliophthora, Myriococcus, Neurospora, Penicillium, Phanerochaete, Phlebia, Pleurotus, Podospora, Polypore, Rhizoctonia, Scytalidium, Pycnoporetus and Tyc.

As used herein, the terms "enzyme protein", "" cellulase preparation "and" endoglucanase preparation "refer to any enzyme product containing at least one endo-glucanase or endoglucanase fusion protein of the invention. Thus, such an enzyme preparation may be a culture medium or a filtrate containing one or more endoglucanases or endoglucanase fusion proteins or one or more endoglucanases or single endoglucanases, a mixture of endoglycanases or endoglucanase fusion proteins and one or more other enzymes. In addition to endoglucanase activity, such a preparation may contain additives such as stabilizers, buffers, preservatives, surfactants, and / or culture medium components.

Preferred additives are those commonly used in enzyme preparations for the application in which the enzyme preparation is used. The enzyme preparation may be in the form of a liquid, powder or granules.

* * 25 '' Consumed culture medium '' as used herein refers to a culture medium of a host comprising the enzymes produced. Preferably, the host cells are separated from said medium after production.

The enzyme preparation may comprise one or more endoglucanases of the present invention, or other cellulase enzymes or endoglucan canase fusion proteins in combination with one or more endoglycanases of the present invention. For example, endoglucanases having, v, different properties can be combined to make an enzyme preparation which is more useful under different conditions.

**: * 'To provide the enzyme preparations of the invention, the host: *.! 35 strains having the desired properties (namely, hosts capable of expressing economically feasible amounts of the endoglucanase enzymes or endoglucanase fusion proteins of the invention) are cultured under appropriate conditions, secreted into the desired enzymes said culture medium by methods known in the art.

The enzyme preparation may comprise, in addition to endoglucanase, one or more ampoules of other enzymes which may be, for example, amylases, lipases, proteases, pectinases and / or oxidases such as laccases and peroxidases. Alternatively, a second enzyme treatment may be performed before, during or after treatment with the endoglucanases of the present invention. For example, the enzyme treatment may comprise one or more Amy-10 glass treatments, one or more cellulase treatments and / or one or more peroxidase and / or laccase treatments. What other enzymes are included in the enzyme preparation or used in the enzyme treatment depends on the application.

The enzyme preparation may be a culture medium with or without native or transformed host cells or may be recovered from the above using methods well known in the art. However, since the endoglucanases of the invention are secreted into the culture medium and exhibit activity under the ambient conditions of the cellulolytic solution, it is an advantage of the invention that the enzyme preparations of the invention can be directly utilized from the culture medium without further purification. If desired, such formulations may be freeze-dried or the enzyme activity may otherwise be concentrated and / or stabilized for storage. The enzyme preparations of the invention are · very economical to obtain and use because (1) the enzymes can be used in their raw form; isolation of specific enzymes from the culture medium is necessary.

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And (2) because the enzymes are secreted into the culture medium, only the culture medium must be recovered in order to obtain the desired enzyme preparations; no:; j ,: no need to extract the enzyme from the hosts. Preferably, the host for such production is Trichoderma, and in particular T. reesei.

The enzyme preparations of the invention may be provided in liquid or solid form, for example, in dried powder or granular or liquid form, in particular in the form of dust-free granules or stabilized liquid. or the enzyme preparation may otherwise be concentrated or stabilized for storage or use. It is contemplated that enzyme preparations containing one or *: * 'more of the cellulases of the invention may be further enriched or deprived of specific enzyme activities, so that they *: * ·: satisfy the requirements for specific uses in a variety of applications. applications, 15 1 1 9325 eg in the textile industry. A mixture of enzyme activities secreted by the host, and in particular by the fungal host, can be selected to be advantageous in a specific industrial application, such as bio-finishing and bio-stone washing.

The enzyme preparations of the invention can be adjusted to meet specific needs in a variety of applications in the textile, detergent, or pulp and paper industries.

Mixtures may be prepared with other macromolecules which may not all be produced in the same host (for example, other enzymes such as endoglucanases, amylases, lipases, proteases, pectinases and / or oxidases such as laccases and peroxidases) or chemicals which may increase potency. or buffering the desired enzyme preparation. Dust-free granules can be coated. Liquid enzyme preparations may be stabilized by the addition of a polyol such as propylene glycol, sugar or sugar alcohol, lactic acid or boric acid, or sodium chloride according to established methods.

Protected forms of the enzymes of the invention may be prepared as described in EP 238 216.

The enzyme preparations of the invention may contain surfactants which may be anionic, nonionic, cationic, amphoteric or a mixture of these types, especially when used as a detergent composition. Useful detergent compositions are described, for example, in WO 94/07998, U.S. Patent Nos. 5,443,750 and 3,664,961.

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If desired, the desired enzyme may be further purified under conventional conditions such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, or the like.

** '. * The enzyme preparations of the present invention are particularly useful in the textile industry, preferably in bio-finishing and bio-stone washing, or in the detergent industry. Other useful areas are in the pulp and paper industry.

• ·: .v 30 “Biofinishing” refers to the use of enzymes in cellulosic fiber containers]: in roll hydrolysis to modify the surface of a fabric or yarn in a manner that v. Permanently prevents purring, improves fabric feel such as softness and smoothness by reducing surface structure linting, **: ** which results in color clarification, improves fabric settling, improves moisture absorption, and can also improve dyeing.

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The enzymatic anti-peeling may be carried out at any stage during the wet treatment of the textile, preferably after removal of the peel and bleaching. The enzymatic process requires equipment with adequate shear forces and mixing, such as a nozzle-gaming machine or washing machine (Nierst-5 rasz V.A. and Warmoeskerken 2003).

Biofinishing is typically performed at a pH of about 4.0 to about 6.0. The reaction temperature may range from about 30 to 70 ° 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 about 20: 1, preferably from 5: 1 to about 10: 1. The treatment time is generally 15 to 90 min, preferably 10 to 30 min. The dosage of enzyme depends greatly 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. Appropriate dosages and conditions will be apparent to those skilled in the art.

The endoglucanases of the invention are particularly useful in the textile industry for the biological finishing of fabrics or garments, e.g., for peeling, linting, color clarification, roughness reduction, various finishes (e.g., "peach-like", "worn out" or "sand") ) and biofinishing yarns (for example, to reduce fluffiness and improve smoothness). The endoglucanases of the present invention and the endo-glucanase fusion proteins described herein can be used in biofinishing in acidic and. under neutral conditions.

*. . ·. The endoglucanases of the present invention and the endoglucanase fusion proteins described herein are useful in detergent compositions for improving the fabric care properties of anti-peeling, graying, color brightening and softening, and textiles. for example, to remove dirt.

There are three steps to stonewashing: removing the glue, rubbing it and finishing. The first step, the peeling process, is normally the first wet treatment of jeans \., T and means starch or other peeling. *! *. removal, which is commonly used for warp yarns to prevent their damage. * · *, during the weaving process. Alpha-amylases are used to remove starch-based paste to obtain an improved and uniform wet-treatment time: V 35. After removal of the paste, the jeans are normally rinsed with water or continued directly with the rubbing step.

17 1 1 9325

The second step, abrasion, can be performed with enzymes or hoh-quartz 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" herein refers to the appearance of the denim fabric when treated with cellulase enzymes or stones or both. As a result of uneven discoloration, there is a contrast between the dyed areas and the dyed areas. Synonyms are "stone-washed appearance" or "worn" appearance. ”In enzymatic stonewashing or biofuel washing, pumice rubbing is completely or partially eliminated and cellulase is added to facilitate the abrasion of Indigo dye from the surface of the fiber. Cellulase treatment can be done using neutral or acidic cellulases or both.

The abrasion is usually followed by a third step, a post-treatment, which includes the washing and rinsing steps, during which detergents, optical brighteners or emollients can be used. After enzymatic treatment, the reaction must be stopped to prevent damage to the treated materials, for example by inactivation of temperature and / or pH, the latter comprising careful rinsing and / or washing of detergent. This ensures that the mechanical strength of the fiber is no longer compromised by the continuous presence of the enzyme.

"Denim", as used herein, refers to denim fabric, generally denim, especially denim. Denim is preferably an indigo dyed denim. Denim can also be treated with indigo, indigo derivatives, or denim treated with some other colors, · '·' · For example an indigo dyed with a sulfur base.

. . ·. Treatment with cellulase (s) can completely replace the treatment with pumice stone. 25 (for example, 1 kg of commercial enzyme equals 100 kg of stones). However, the pulp • · · j * V treatment can be combined with the pumice treatment when it is desired to produce :: i.! heavily worn finish. The peach-like effect of creating a thin layer of hairs protruding from the surface is also achieved by washing combining neutral cellulases with pumice stones. The cellulases of this invention are particularly useful for a worn-out appearance. and minimize back discoloration in biofuel washing.

.V. Typically, biofuel washing is carried out at a pH of about 3.0 to about 8.0, and preferably at a pH of about 4.0 to about 6.0. The reaction temperature may range from about 30 ° C to about 70 ° C and is preferably about 50 ° C to about 60 ° C. Solution ratio (volume ratio of liquid to fabric

·· I

ß V 35 per weight) may range from about 3: 1 to about 20: 1, preferably 5: 1 to about 10: 1. The treatment time * "*! May vary from 15 min to 90 min and preferably from 30 min to 60 min. It must be emphasized that the enzyme dose 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 endoglucanases or endo-5 glucanase fusion proteins, and the required enzyme dose will then be substantially lower.

The textile material to be treated with the enzyme preparations of the invention may be made from natural cellulose containing fiber 10, or artificial cellulose containing fibers, or mixtures thereof. Examples of natural cellulosic fibers are cotton, plain, hemp, jute and ram. Examples of artificial cellulose fibers include viscose, cellulose acetate, cellulose triacetate, rayon, cupro and lyocell. The above-mentioned cellulosic fibers can also be used as blends of synthetic fibers, such as polyester-15 terry, polyamide or acrylic fibers. The textile material may be yarn or knitted or woven, or otherwise formed.

The endoglucanases of the invention and the endoglucanase fusion proteins described herein, besides being particularly useful for treating fabrics, are generally useful in any region that requires cellulase activity.

In the pulp and paper industry, cellulases can be used: for example, for fiber removal of various recycled papers and boards having a neutral · * · * · or alkaline pH, or for fiber modification. ·, Improving or adding dewatering to paper production. Other · *] *. The examples include removal of printing paste thickener and excess dye after · · · · · · Textile printing and processing for animal feed. For example, if V: the intended application is to improve the strength of the mechanical pulp, then the enzyme preparations of the invention can provide one or more of these proteins by increasing or facilitating the binding capacity of cellulosic fibers. Similarly, in the application of fiber pulp processing, the endoglucanases of the invention and the endoglucanase fusion protein prepared, v. The teas can provide one or more of these proteins at a level that increases or facilitates such swelling.

The endoglucanases of the invention and the endo- ·· ·: V 35 glucanase fusion proteins also described herein provide unexpected advantages when used in the textile industry and especially in bio-finishing such as anti-pitting and stone washing. The endoglucanases of the invention and the described endoglucanase fusion proteins are significantly more effective than the cellulases of the prior art. At least four times lower doses could be used in bio-finishing. In other words, higher potency is achieved using the endoglucanases and endoglucanase fusion proteins of the present invention. In the prevention of clumping, the endoglucanases of the invention and said endoglucanase fusion proteins were more effective and produced a stable smooth surface.

The invention will be described in more detail in the following examples, which are not to be construed as limiting the scope of the invention but merely for clarifying the use of the invention.

Example 1. Cultivation of Acremonlum thermophilum ALK04245

Acremonium thermophilum strain ALK04245 was grown in a 2-liter bioreactor (Braun Biostat® B, Braun, Melsungen, Germany) in the following woman, g / l: Solka Floc cellulose 40, cornmeal 15, used mash 5, oat and wheat xylan 3 , locust bean powder 3, (NH 4) 2 SO 4 5 and KH 2 PO 4 5. pH range 5.2 ± 0.2 (NH 3 / H 2 SO 4), aeration 1 vm, mixing 300-600 rpm, antifoam control with Struktol® and temperature 42 ° C. The culture time was 4 days. After culture, cells and other solids were harvested by centrifugation and the supernatant was collected.

• * »i«

EXAMPLE 2. Purification of Endoglucanase from Acremonium thermophilum v: ALK04245 The culture supernatant of Acremonium therrmophi- lum ALK04245 grown as described in Example 1 was incubated at 70 ° C for 24 hours. after that it was concentrated by ultrafiltration. Pure endoglucanase was obtained by sequential purification by hydrophobic interaction and cation exchange chromatography and subsequent gel filtration. The endoglucanase activity of the fractions collected during purification was determined using carboxymethyl. stylized cellulose (CMC) as a substrate (according to IUPAC Procedure 1987).

The concentrated culture supernatant was placed on a HiPrep 16/10 Butyl FF hydro v *: phobic interaction column (GE Healthcare) equilibrated in 20 mM potassium phosphate buffer, pH 6.0 containing 1 M (NH 4) 2 SO 4. Commitment: v. These proteins were eluted with a linear gradient from yeast buffer to 5 mM potassium phosphate, pH 6.0. Fractions were collected and endoglucanase activity was determined as described above. Endoglucanase activity eluted over a wide conductivity range of 120-15 mS / cm.

The combined fractions were loaded on a HiTrap SP XL cation exchange column (GE Healthcare) equilibrated with 8 mM sodium acetate, pH 4.5. Si-5-bound proteins were eluted with a linear gradient of 0-0.25 M NaCl in equilibration buffer. Proteins with endoglucanase activity eluted at a conductivity range of 3-7 mS / cm. Cation exchange chromatography was repeated and the protein eluate was concentrated by freeze-drying.

The dissolved sample was loaded onto Superdex 75 HR10 / 30 gel filtration column 10 (Pharmacia) equilibrated with 20 mM sodium phosphate buffer, pH 7.0 containing 0.15 M NaCl. The main protein fraction eluted from the column with a retention volume of 13.3 ml. The protein eluate was pure as determined by SDS-polyacrylamide gel electrophoresis and the molecular weight was estimated to be 40 kDa. The specific activity of the purified protein, designated Acremonium thermophi-15 iumvn EG_40 or At EG_40 (SEQ ID NO: 2), at 50 ° C was determined to be 450 nkat / mg (according to IUPAC Procedure 1987) using CMC: aa as a substrate).

The thermal stability of the purified endoglucanase was determined at various temperatures. The reaction was performed in the presence of 0.1 mg / ml BSA at pH 20 5.0 for 60 min using CMC as substrate. At EG_40 was stable up to 80 ° C. The reference enzymes EGI (Cel7B) and EGH (Cel5A): t. Teesei retained 100% of their activity up to 60 ° C and 65 ° C, respectively.

. , \ The purified Acremonium ther- · "·, 25 mophilum ALK04245 EG_40 protein (SEQ ID NO: 2) was first alkylated and digested into tryptic peptides. The generated peptides were desalted and * :: 7 partially separated. internal peptides • «* ··· 'was sequenced by electrospray ionization combined with a tandem mass spectrometer (ESI-MS / MS) using a Q-TOF1 (Waters Micromass®) .Available: 30 dut internal peptide sequences are listed in the table. 1.

·· • · * · ··· • · · · »1« • · IM • 1 • «

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Table 1. Internal peptide sequences determined from Acremonium thermophilum EG_40 cellulase _Peptide__S SEQ ID NO:

Peptide 1__QSCSSFPAPLKPGCQWR__5_

Peptide 2__YALTFNSGPVAGK__6_

Peptide 3__VQPSELTSR__7_

Peptide 4__NQPVFSCSADWQR__8_

Peptide 5__YWDCCKPSCGWPGK__9_

Peptide 6 PTFT 10

Example 3. Cloning of the ce / 45A and ce / 45B genes of Acremonium thermophilum (ALK04245) The basic methods of molecular biology were used to isolate DNA (plasmids, DNA fragments) and enzyme treatments, E. coli transformations, etc. The basic methods used are described in molecular biology. Sambrook, J., et al., 1989 and Sambrook J. and Russell, DW, 2001.

The genomic library of Acremonium thermophilum ALK04245 was constructed in the Lambda DASH®II 'vector (Stratagene, USA) according to the manufacturer's instructions. Knomosomal DNA isolated by the method of Reader and Brodan, 1985 was partially digested with Sau3A. The digested DNA was size-fractionated on an agarose gel and fragments of the selected size (about 5-23 kb) were isolated, defos-. was lyophilized and ligated into BamHI digested lambda vector dwarf arms. Liga- • · · *. The mixture was packaged using Gigapack III Gold packaging extracts according to the manufacturer's instructions (Stratagene, USA). The titer of the genomic library was 3.7 x 10 5 pfu / ml and the amplified library was 4.2 x 10 8 pfu / ml.

ί.ί: The internal peptide sequence purified Acremonium thermophi ··· Lumin EG_40 cellulase obtained as described in Example 2 had 20 homologies to glycosyl hydrolase family 45 cellulases such as Thiela-: V: via terrestris endoglucanase (GenBank82). *** · Cel45A Cellulase (GenBank Accession No. · ·· AJ515703) from Melanocarpus aibomyces. To amplify the probe to screen for the gene encoding A. thermophilum EG 40 ee · (ce / 45A; SEQ ID NO: 1), degenerate primers were designed based on the peptide sequences listed in Table 1 (Example 2). The order of the peptides in the protein sequence ···· and the corresponding sense and antisense character of the primers were deduced from the comparison with the homologous Cel45A sequence of M. aibomyces. Sense primer 22 119325 (TAYTGGGAYTGYTGYAARCC, SEQ ID NO: 11) is based on amino acids 1-6 of peptide 5 (SEQ ID NO: 9) and the antisense primer (RTTRTCNGCRTTYTGRAACCA, SEQ ID NO: 12) is based on the homologous M. albomycesin CelinN peptide W45N peptide; ). The PCR reaction mixtures contained 50 mM Tris-HCl, pH 9.0.15 mM (NH 4 SO 2, 0.1% Triton X-100, 1.5 mM MgCl 2, 1 mM dNTPs, 0.5 pg each primer, 1 unit Dynazyme EXT DNA polymerase (Finnzymes, Finland) and approximately 0.5 pg Acremonium genomic DNA The conditions for the PCR reactions were as follows: denaturation at 95 ° C followed by 30 1 minute cycles at 95 ° C, 1 minute 10 primer attachment step at 50-60 ° C, 2 minute primer extension step at 72 ° C, and final elongation step at 72 ° C 10 The extension products were screened on an agarose gel.

Two PCR products were obtained from the Acremonium PCR reaction. DNA fragments of approximately 0.6 kb (SEQ ID NO: 14) and 0.8 kb (SEQ ID NO: 15) were isolated from an agarose gel and cloned into the pCR4-TOPO®TA vector (Invitrogen, USA), resulting in plasmids. pALK1710 and pALK1711, respectively. The cloned PCR products were characterized by sequencing and performing Southern blot hybridization (as described below) on genomic Acremonium DNA digested with several restriction enzymes. Hydrogenation patterns obtained with two fragmen-20 accounts under strong washing conditions suggest that two putative endoglucanase genes could be screened from the Acremonium: * ·. · Genomic library. The deduced amino acid sequences of both PCR products have homology to several published endoglucanase sequences of the glycosyl hydrolase family (BLAST program, National Center for Biotechnology Information; Altschul et al. 1990).

i ".: Insert from plasmid pALK1710 and pALK1711 were isolated by restriction enzyme digestion and labeled with digoxigenin according to the manufacturer's instructions * ··· * (Roche, Germany). Approximately 1-2x105 plaques from the amplified Acremonium genomic library were transferred to a nitrocellulose filter and Hybridization temperature was 68 ° C and the filters were washed 2x5 minutes at room temperature using 2xSSC-0.1% SDS followed by 2x15 minutes at 68 ° C. 0.1 x SSC - 0.1% SDS Several positive plaques were obtained, of which five highly hybridizing plaques were purified from both screens Phage DNA j * V 35 was isolated and analyzed by Southern blot hybridization. *: · *: Restriction fragments of phage DNAs were subcloned into pBluescript II KS + vector 23 119325 (Stratagene, USA) and the essential parts In both cases, the subcloned phage fragment contains the full-length gene of interest.

Table 2 summarizes data from probes used for screening endoglucanase genes, selected phage clones containing restriction fragments containing full-length genes with their promoter and terminator regions, plasmid names containing subcloned fusion fragments, culture collection (DSM) for E. coli strains carrying these 10 plasmids. The deposits were made under the Budapest Treaty on 13 May 2005.

Table 2. Probes used for cloning endoglucanase genes, selected phage clones and subclones, plasmid names and corresponding accession number for E. coli strains

Gene Genomic Screening Phage Clone Subcloned Plasmid E. coli Library Used Test Fragment Depository _______mero

At A. thermo- pALK1710 P24 5.5 kb Smal pALK1908 DSM

ce / 45A philum 17324 __ALKQ4245______

: At A. thermo- pALK1711 P41 6,0kbX /? Ol pALK1904 DSM

ce / 45B philum 17323 __ALKQ4245_______

• · ·> 11 II I I I ............- -lM I. ... 1 I

Γ 15 The two genes named Atce / 45A (SEQ ID NO: 1) and> »t

At ce / 45B (SEQ ID NO: 3), the relevant data are summarized in Table 3 and the relevant data for the respective derived protein sequences, At EG_40 (SEQ ID NO: 2) and At EG_40_like (SEQ ID NO: 4), are as follows: Purified: V: Acremoniumm EG_40 endoglucanase peptide sequences were detected in the corresponding deduced amino acid sequence of the cloned 20 gene, confirming that the appropriate gene was cloned.

The full-length Ate / 45A gene (SEQ ID NO: 1) is 1076 bp long, cleaved by two 59 bp and 123 bp introns and encodes a 297 amino acid po; lipid At EG_40 (SEQ ID NO: 2). The putative signal peptide cleavage site *: ** is after Ala21 and the mature protein N-Terminus starts with Leu22 with the mature 24 119325 protein (containing CBD) comprising the amino acids 22-297 of SEQ ID NO: 2. is a C-terminal consistent cellulose binding domain consisting of the full length polypeptide amino acids Lys265 - Leu297. The predicted mature protein after signal peptide cleavage 5 has a molecular weight and a pI of 28625 Da and 4.79, respectively (prediction made using the Compute pI / MW tool on the ExPASy server, Gas-claimser et al., 2003). The protein has two putative N-glycosylation sites N-X-S / T (predicted using NetNGIyc 1.0, Gupta et al., 2004).

Similarly, the full-length at ce / 45B gene (SEQ ID NO: 3) is 1013 bp 10 long, cut by two 155 bp and 102 bp introns, and encodes the 251 amino acid polypeptide At EG_40_like (SEQ ID NO: 4). The putative signal peptide is cleaved after Ala20 and the mature protein has an N-Terminus starting with Gln21, the mature protein comprising amino acids 21 · 251 of SEQ ID NO: 4. EG_40_like cellulase does not have a C-terminal uniform cellulose binding site. The predicted mature protein after cleavage of the signal peptide has a molecular weight and a pI of 23972 Da and 6.11, respectively (prediction made using the Compute pI / MW tool on the ExPASy server, Gasteiger et al., 2003). The protein has two putative N-glycosylation sites N-X-S / T (predicted using NetNGIyc 1.0, Gupta et al., 20 2004).

. . Table 3. Summary of endo- .1 2 3 4. / glucanase genes isolated from Acremonium thermophilum ALK04245 • · • · · • n- _: es1s Endoglucan-Length intro- Coding of Introns Introns Sequence: number of naase genes (bp) length no sa (bp) {b (bp) · 1 · 1 (a • · · \ • · ^ 5 _ _ __

At ce / 45A 1076__891 2__59,123 1_

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119325 The derived protein sequences of A. thermophilum EG_40 and EG_40Jike cellulases are similar to the 45 cellulases of the glycosyl hydrolase family (Table 5). The closest sequence homologies observed for EG_40 / Cel45A and EG_40_like / Cel45B were the endoglucanase sequences in Thieiavia terrestris (GenBank accession number 5 CQ827970) and Myceliophthora thermophila (GenBank accession number AR094305). The alignment was performed using the Needle program of the EMBOSS program package.

Table 5. Comparison of Derived Protein Sequences of Acremonium thermophilum EG_40 and EG40_like Cellulases with Their Homologous Antibodies

Organism, enzyme and deposit number__Identity (%)

Acremonium thermophilumin EG_40

Thielavia terrestisin EG45, CQ827970 77.3

Melanocarpus albomycesin Cel45, AJ515703 75.3

Neurospora in the spindle, hypothetical XMJ324477 68.9

Humicola grisea var thermoidea, EGL3, AB003107 67.5

Humicola insolensin EG5, A23635 67.3

Myceliophtora thermophila family 45, AR094305 57.9

Acremonium thermophilumin EG_40Jike__53.7

Acremonium thermophilumin EG_40Jike: · **: Myceliophtora thermophila family 45, AR094305 66.9: Magnaporthe grisean 70-15 hypothetical, XM_363402 61.9

Thielavia terrestisin EG45, CQ827970 56.8: Acremonium thermophilumin EG_40 53.7 • · *: Melanocarpus albomycesin Cel45, AJ515703 52.8 · · 1 1 ***** Example 4. Acremonium thermophilunmn EG_40 and EG_40_like-cellulose. glass production in Trichoderma reesees • ♦ • · ·

Expression plasmids were constructed for the production of recombinant A. thermophilumin: ···: EG 40 / Cel45A and EG 40 like / Cel45B cellulases. Both 15 genes (ce / 45A or ce / 45B) including their own signal sequences are fused. was precisely inserted into the T. reesei cbhf promoter (ce17A) by PCR (Table 6).

./.* cb / 7 f promoter, cb / j terminator and amdS marker gene were included as described in Paloheimo et al., 2003. The linear expression cassette (Figure 1) was isolated from the vector backbone by restriction enzyme digestion, 27119325 were transformed into T. reesei A96 and the transformants were selected with 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 modifications as described in Karhunen et al., 1993. Transformants were purified on a selection of plates through individual conidia before being sprouted with potato extract.

Table 6. Expression cassettes constructed for production of Acremonium thermophilum EG40 and EG40_like cellulases in Trichoderma reese. The schematic structure of the Eks-10 press cartridges is illustrated in Figure 1

Endoglucanase Expression Plasmid Expression Cassette Heterologic ___ size (a__terminator (b

At EG_40 pALK1920 10.9kbNotl 156 bp ____ (HindUl)

At EG_40_like pALK1921 8.6 kb EcoRI 282 bp (Sspl) (a) The expression cassette for T. reesei transformation was isolated from the vector backbone by Eco RI or / Vof1 digestion (b Nucleotides after the STOP codon of the cloned gene included in the expression cassette. used in the ex 15 constructs of the press cassette, in the 3 'region is indicated in brackets i «« * ·· • · ··· V: The endoglucanase production of transformants was analyzed from culture supernatants of flask flasks (50 ml) grown in shakers for 7 days in complex cellulase expression. - in 20 media (Joutsjoki et al. 1993) buffered with 5%: ***; H 2 O PCMI at pH 5.5 The enzymatic activity of the recombinant protein was measured by the release of reduced sugars from the culture supernatant from carboxymethyl cellulose (2%). CMC) at 50 ° C in 50 mM citrate buffer pH 4.8 · · · substantially k uten are described in Bailey, M. J. and Nevalainen, K. M. H., * Γ * 1981; Haakana, H., et al., 2004. Recombinant protein production was also detected from culture supernatant by SDS-polyacrylamide gel electrophoresis. EG_40-:]]]: specific polyclonal antibodies were produced in rabbits (University of Helsinki, • v, Finland). EG_40 cellulase expression was verified by Westem blot analysis with anti-EG_40 antibodies using the ProtoBlot Western blot AP system (Pro-♦ 28 119325 mega). Genotypes of selected transformants were analyzed by Southem blotting using an expression cassette as a probe.

The pH optimum of the heterologously produced EG_40 / Cel45A and EG_40_like / Cel45B cellulases was determined in Universal Mcllvane buffer pH-5 between 4.0 and 8.0 using CMC as substrate. As shown in Figure 2A, the pH range of EG_40 / Cel45A cellulase is relatively wide (4.5 to 6.0) with optimum pH 5.5. The pH optimum of EG_40_like / Cel45B was determined to be pH 5.0-5.5. The optimum temperature for the enzyme activity of EG_40 / Cel45A and EG_40_like / Cel45B cellulases was determined to be 75-80 ° C and 60 ° C in said order (Figure 2B). The thermal stability of the heterologously produced EG_40 / Cel45A cellulase is comparable to that of the purified protein.

Selected transformants RF6118 (At EG_40) and RF6071 (At EG_40_like) were cultured in a 2-liter bioreactor for four days (28 ° C, pH 4.2) to obtain material for application tests (see Examples 7-10).

Example 5. Production of EG_40 cellulases of Acremonium thermophilum ALK04245 lacking a cellulose binding domain or a cellulose binding domain and a junction region

Two ce / 45A deletion constructs are made to produce EG_40 cellulases of Acremonium thermophilum ALK04245 lacking the cellulose binding domain (CBD), At cel45A_CBDIess, or CBD yn-20 junction region, At cel45A_linkerCBDIess; the former lacks the area encoding the CBD and the function ·: *! it also lacks a linking region between the catalytic core and the cellulose binding domain.

• · ·. * “Basic methods of molecular biology are used as described in the precursors: 25 at 3. The 3 'end of the ce / 45A gene is confirmed by PCR. The antisense primer is designed to exclude a CBD or splice + CBD region product and the PCR product is ligated to the 5 'fragment of the ce / 45A gene to rebuild full-length genes (SEQ ID NOs: 16 and 18, respectively). . : V: The expression plasmids for the production of A. thermophilum EG_40 / Cel45A cellulase CBDIess: ***: 30 and linkerCBDIess variants are constructed and recombinant proteins (SEQ ID NOs: 17 and 19, respectively) are produced in Trichoderase *, such as is described in Example 4.

• · • · M »·» Φ · * • · • • • • 29 119325

Example 6. Production of recombinant Acremonium thermophilum ALK04245 EG_40_like + CBD fusion protein

To produce the EG_40_like + CBD fusion protein of recombinant Acremonium thermophilum ALK04245 (SEQ ID NO: 21), the cellulose binding domain (CBD) of EG_40 / Cel45A cellulase is ligated to EG_40Jike-seluase. The construct contains the catalytic domain of EG_40_like (amino acids 1-242 of the full length polypeptide) linked to the EG_40 cellulase junction region and CBD (amino acids 235-297 of the full length polypeptide).

Basic molecular biology techniques are used as described in Example 3. First, a unique A / rul restriction site near the C-terminal end of the EG_40_like sequence is introduced by PCR. This allows direct fusion of any blunt-ended DNA after the amino acid S242 of the EG_40_like polypeptide. The + CBD region of the EG_40 coding gene (ce / 45A) was confirmed by PCR and its restriction fragment was ligated into the ce / 45B gene 15 (after S242) to generate At ce / 45B_ce / 45A splice CBD (SEQ ID NO: 20).

The expression plasmid EG_40_likeCBD for cellulase production is constructed and the recombinant protein (SEQ ID NO: 21) is produced in Trichoderma as described in Example 4.

Example 7. Efficacy of EG_40 cellulase preparation in denim finishing at various temperatures

Acremonium thermophilum EG_40 cellulase from strain RF6118, produced using Trichoderma reese as host, as described in Ex. ·. cat 4, was tested for its ability to create a similar worn appearance that • «· ': **. ·, obtained by pumice stone washing in denim at different temperatures. A commercial V 25 EGII-enriched preparation produced using Trichoderma as host V .: (US 5,874,293), which is effective in denim finishing, was used for comparative bone at 50 ° C.

Jeans made from Indigo-stained denim twill were used as a test mockup after removal of the adhesive with ECOSTONE A200 alpha-amylase. Cellulase treatments were performed on an Electrolux Wascator FOM 71 CLS washing machine under the conditions described in Table 7.

• '·' V 'EGII-enriched stabilized enzyme concentrate was dosed at V * 0.23% by weight of fabric, a typical dose formulation for industrial: V applications. The concentrated and stabilized EG_40 preparation obtained from pilot *: **: 35 mentions was dosed at 0.18%. When calculated as [protein] relative to the protein content, the doses were about 0.20 mg and 0.035 mg per gram of fabric using Bio-Rad Protein Assay Dye Reagent (BioRad, Hercules, CA, USA) and bovine gamma globulin as standard. After dehydration, the cellulase enzyme was inactivated by raising the pH above 11 with 5 g of NaOH by the addition of 5 (10 min, 40 ° C) and rinsing three times. The jeans were 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 / 2 °). The color of the right and wrong sides of the denim 10 was measured after the removal of the adhesive (i.e. before the cellulase treatment) and after the cellulase treatment. Each measured value on the right side of the denim was approximately the average of 40 measurements. Two pairs of jeans were used in each test and the final result was their mean. The results are shown in Tables 8 and Figure 3.

Table 7. Test Conditions / Process-15 Parameters Used for Cellulase Treatments

Prosessiparametri__

Denim load__1.3-1.4 kg_

Water 19 liters_ pH control (pH 5-5.3) __ 5 ml acetic acid (80%) _. , Time__45 min_ Temperature__40, 50.60 or 70 ° C_ * · * / Cellulose Dose_I 0.18% or 0.23% by weight of fabric • · · · · · · · · · · · · · · · · · · · · · · · · · ·

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The results in Table 8 and Figure 3 show that the stone-washing effect of EG_40 was very good at a low dose interval. For strain RF6118, a similar level of abrasion (brightness L12) was obtained at 50 ° C with a 6-fold lower protein content compared to the EGII-enriched preparation.

Example 8. Enhancement of the Washing Effect of EGII-Enzyme Enzyme Preparation on EG_40 Cellulase in Denim Enhancement The EGII-enriched preparation was enhanced with EG_40 (as in Example 7) and the enhanced preparation was compared to EGII-enriched preparation in denim biofilm washing. The denim and the test system for biofuel washing were similar to the pre-10 brands 7 except at 50 ° C. The effect of the cellulase treatment was also evaluated as in Example 7. Enzyme preparations were dosed at 3-5 grams, resulting in 0.22-0.38% fabric weight (Table 9).

The results in Table 9 and Figure 4 show that EG_40 can also be used to improve the abrasion effect of an EGII-enriched preparation. The EGII-enriched preparation alone had similar levels of brightness to those obtained with a mixture of 70% EGII-enriched concentrate and 30% EG_40 cellulase concentrate, even with the added dose.

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Example 9. Efficacy of EG_40_like cellulase preparation in denim finishing

The EG_40_like fermentation fluid produced from Trichoderma reese as a host as described in Example 4 was compared to 5 EGII-enriched concentrates in denim biofilm washing. The denim and the test system for biofuel washing were similar to Example 7 except for the temperature of 60 ° C and the amount of denim equalized to 1430 g with an additional piece of denim not included in the measurements. The effect of the cellulase treatment was also evaluated as in Example 7.

The results in Table 10 show that the abrasion level of EG_40_like was obtained with less back dyeing (indigo dye repainting) on the wrong side of the denim. In particular, the pockets had a higher brightness and were less blue.

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Example 10. Efficacy of EG_40 and EG_40 Enhanced EGII Enriched Preparation in Biofinishing (Anti-Peeling) used in bio finishing formulations. Cellulase treatments were performed on an Electrolux Wascator FOM 71 CLS washing machine under the conditions described in Table 11.

Two types of low quality blue polo collar knit shirts with fluffy surface made of 100% jersey-based cotton fabric or elastic made from 95% cotton and 5% spandex were used as test material with padding. Samples were first pre-washed for 10 minutes at 60 ° C with 1 ml / L surfactants / wetting agents (San-doclean PCJ Sandos and Imacol CN from Clariant) and rinsed 3 times. 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. The enzyme was inactivated as described in Example 7, except at a temperature of 60 ° C during alkaline rinsing, and pieces of knitwear were rinsed three times and dried in a tumble dryer.

Table 11. Test Conditions / Process Parameters Used in Bio-Finishing Treatments • ·

• # Μ ........ IMI Il I II IW II ·· III I - MH

] · Ϊ ·: Process Parameter__: 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 • Λ '............................. - I m mm—

· *** · Temperature at 60 ° C

··· '"'" "" /.e Cellulose Dose_0.04% - 0.63% by weight of fabric • ·•••••••••••••••••••••••••• ···· • · 37 119325

The effect of the cellulase treatment was assessed visually with the naked eye and magnifying glass. Prewashed sample Without enzyme was used as a control. The results are shown in Table 12 and the digital camera images taken with the macro lens are shown in Figure 5.

EG_40 and EG_40-enhanced EGII-enriched preparation had excellent anti-peeling properties compared to the commercial EGII-enriched formulation used in the typical dosing interval for this enzyme concentrate in a biofinishing application. EG_40 at least 8 times lower dose and EGII-enriched EG_40 at least 4 times lower doses than EGII to achieve similar effect,

Protein levels in bioreactor culture supernatants are somewhat lower for the RF6118 strain than for the Trichodarma producer strain of the EGII-enriched preparation when analyzed by the assay for the protein used. Nevertheless, the culture medium EG_40 is at least 4-6 times more volumetric in bio-finishing.

• t • · · · * · · · M «M M M M M M M M M M M M M M M M * M M · * • * I * · * · · • • • • • • • • · · · · · · · · · · · · · · · · · · · ··· · «« · • · • «* ·· *« * · 119325 38

Table 12. Results of bio-finishing treatments with EG_40- and EG_40-enhanced EGII-enriched preparations as compared to EGII-enriched alone.

Sample Dose Dose, Anti-Bumping Protein g% owf * 'b) mg / g Carrier EGII-Enriched Concentrate 6.3 0.63 +++++ 0.55 Radium_____ EGII-Enriched Concentrate 3.2 0.32 +++ 0.27 carat_____ EGII enriched + EG_40- 3.2 0.32 +++++ 0.21 mixture 70% + 13% _____ EGII enriched + EG_40-1.6 0.16 ++++ + 0.10 mixture 70% + 13% _____ EGII enriched + EG_40-0.8 0.08 +++ 0.052 mixture 70% + 13% _____ EG40 concentrate, 1.6 0.16 +++++ 0.050 RF6118_____ EG_40 Concentrate, 0.8 0.08 +++++ 0.025 RF6118_____ · * ·, · EG_40 Concentrate, 0.4 0.04 +++ 0.012 RF6118______, Prewashed only, - • «* enzyme • · *.

| * V by weight of fabric :: i .: b) +++++ Excellent anti-peeling effect, visually very clean • e * ··· * surface +++ Good anti-peeling effect, visually relatively clean surface \ v - Dense surface peeling / and or severe nausea • • te ·••••••••••••••••••••••••••••••••••••••••••••••••• 39

List of deposited organisms

Acremonium thermophilum ALK04245 was deposited with Centralbureau Voor Schimmelcultures at Uppsalaala 8, 3584 CT, Ultrecht, The Netherlands (CBS) on September 20, 2004 under the number CBS 116240.

Escherichia coli containing plasmid pALK1908 was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, May 13, 2005 under DSM 17324.

Escherichia coli containing plasmid pALK1904 was deposited with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, May 13, 2005 under DSM 17323.

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• · 119325 42

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2052060EN.ST25.txt SEQUENCE LISTING

<110> AB Enzymes GmbH <120> Novel Enzymes <130> 2052060FI

<160> 21 <170> Patent Version 3.1 <210> 1 <211> 2334

<212> DNA

<213> Acremonium thermophilum <220> <221> misc_feature <222> C13) .. C13) <223> n means a, t, c, or g • · · ♦ ft * • ft «<· • · ·

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• · | *! 1 <222> (715) .. (797) »» ·! · '· <223> • i * • · • · · <220> • · • ft · * ·' · * <221> intron ··· ft · * ·; · * <222> (798) .. (856) sV: <223> ft · ft • · • ft • ft * • <220> ft ft.

....: <221> CDS

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<221> CDS

<222> (1229) .. (1787) <223> <400> 1 tctgtctctt gtntcagaac agatctcctg gcggcctgct ttgccggtcc gaattgcgat 60 cgatgcaacg tcgattgcat acgagctaag cccgtctcgt gataaccgca aggggtcttc 120 cgagtttctg tctgcgaccc aggcattttc cgatttgtgt gcggggaccc aactgtcttc 180 tggggagtac ctggtgacaa aagcacagat aaacagatgg atgacggtat tgctgtgata 240 tcgccgtggc gctgaatcct ttctcttcgc taccaagata tttattcccc gttgtgaaat 300 cttctattca gcccatccca tccggcaaca cgcatctgct tttcgttccg gcattccgat 360 acctggttcc tggagtgcct accgagcctc gcttcctggg atcgggcgtt gcaccccgcc! aaaccctatg ccccaaacgg tacggacaag gatgccggac cccggttttg tccagaaagg 480; 1 · '· ttgcattcct acccacctcg ctggagccac aacatgcaga tcaccgcccg agggaggaca 540 tgtgtggtgc agggacgttg gcaactctgc tgtgtctgaa gtatatgagg ccgatggttc 600 · · · · · 2 tccttgcaca aagcagagaa tggagtagcc agctcctcct caccagagtc gcctttgcag 660 · »· · · • 1 · cgtctcggca ttgcaggctc cccatcgtca gcatttcact tctcagcaac gaac atm 717 · 2 · · Met:: 1 · »· cgc tee tea ccc ttt etc cgc gca get ctg get gee get ctg eet ctg 765

Arg Ser Ser Pro Phe Leu Arg Ala Ala Leu Ala Ala Ala Leu Pro Leu • Y: 5 10 15 • · · 3 · age gee cat gee etc gac gga aag teg time ag gtatgccaat cctcgtacct 817 ··· Ser Ala His Ala Leu Asp Gly Lys Ser Thr Arg Λ 20 25 • · · ♦ · ·! ·> Ctgccctctg tagaaacaag tgaccgactg caaagacag a tae tgg gac tgc tgc 872 t! Tyr Trp Asp Cys Cys I 30 2 · • · · 3 * · 'aag ccg tee tgc ggc tgg ccg gga aag gee teg gtg aac cag ccc gtc 920 ···· ♦ Lys Pro Ser Cys Gly Trp Pro Gly Lys Ala Ser val. Asn Gin Pro val * 1 35 40 45 page 2 2052060Fl.ST25.txt 119325 ttc teg tgc teg gee gac tgg cag egc ate age gac ttc aac geg aag 968

Phe ser cys ser Ala Asp Trp Gin Arg lie Ser Asp Phe Asn Ala Lys 50 55 60 65 teg ggc tgc gac gga ggc tee gee tae teg tgc gee gac cag time ccc 1016

Ser Gly Cys Asp Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gin Thr Pro 70 75 80 tgg geg gtc aac gac aac ttc teg tae ggc ttc gca gee aeg gee ate 1064

Trp Ala val Asn Asp Asn Phe ser Tyr Gly Phe Ala Ala Thr Ala lie 85 90 95 gee ggc ggc tee gag tee age tgg tgc tgc gee tgc tat gc 1105

Ala Gly Glu Gly Ser Ser Ser Trp cys Cys Ala Cys Tyr Ala 100 105 110 gtgagttctc tgcaagccgc ttcccacccc cgctttctgt gcaggccgct tcccccctac 1165 ccacccactt cccccccccc gcctctgtga tcgggcatcc gagetaagtt 1225 gcgtgtcgtc a cag acc ttc aac etc teg gtc ccc ggc ggc aag GEG ace atg gtg gtg 1274

Leu Thr Phe Asn Ser Gly Pro val Ala Gly Lys Thr Met Val Val 115 120 125 cag teg acc age acc ggc ggc gac ctg ggc age aac cag ttc gac etc 1322

Gin ser Thr ser Thr Gly Gly Asp Leu Gly Ser Asn Gin Phe Asp Leu 130 135 140 gee ate ccc ggc ggc ggc gtg ggc ate ttc aac ggc tgc gee tee cag 1370

Ala lie pro Gly Gly Gly val Gly lie Phe Asn Gly Cys Ala Ser Gin 145 150 155 ttc ggc ggc etc ccc ggc gee cag tae ggc ggc ate age gac ege age 1418

Phe Gly Gly Leu Pro Gly Ala Gin Tyr Gly Gly lie Ser Asp Arg Ser 160 165 170 cag tgc teg tee ttc ccc geg ccg etc cag ccg ggc tgc cag tgg ege 1466

Gin Cys Ser Ser Phe Pro Ala Pro Leu Gin Pro Gly Cys Gin Trp Arg 175 180 185 190. ·. : ttc gac tgg ttc cag aac gee gac aac ccc acc ttc acc ttc cag ege 1514 *. *: Phe Asp Trp Phe Gin Asn Ala Asp Asn pro Thr Phe Thr Phe Gin Arg 195 200 205 • «·. gtg cag tgc ccg tee gag etc time tee ege time ggc tgt aag ege gac 1562 val Gin Cys Pro Ser Glu Leu Thr Ser Arg Thr Gly Cys Lys Arg Asp: 210 215 220 • · · ··· ·:. *. gac gac gee age tat ccc gtc ttc aac ccg cct age ggt ggc tee ccc 1610:.::: Asp Asp Ala Ser Tyr Pro val Phe Asn Pro Pro ser Gly Gly Ser Pro 225 230 235 • • · · age acc acc age acc acc. acc age tee ccg tee ggt ccc aeg ggc aac 1658 ser Thr Thr ser Thr Thr Thr Ser ser Pro Ser Gly Pro Thr Gly Asn 240 245 250 • · · • ·. ***. cct cct gga ggc ggt ggc tgc act gee cag aag tgg gee cag tgc ggc 1706 ... Pro Pro Gly Gly Gly Gly cys Thr Ala Gin Lys Trp Ala Gin Cys Gly 255 260 265 270 * * * • · · 99c act 99c ttc ac9 99c tgc acc acc tgc gtc teg ggc acc acc tgc 1754:: Gly Thr Gly Phe Thr Gly Cys Thr Thr Cys Val ser Gly Thr Thr Cys: * 275 280 285 • · · • * ·:. * cag gtg cag aac cag tgg tat tee cag tgt ctg tgagcgggag ggttgttggg 1807 ....: Gin Val Gin Asn Gin Trp Tyr Ser Gin Cys Leu * * 290 295

Page 3 119325 2052060FI.ST25.txt gtccgtttcc ctagggctga ggctgacgtg aactgggtcc tcttgtccgc cccatcacgg 1867 gttcgtattc gcgcgcttag ggagaggagg atgcagtttg agggggccac attttgaggg 1927 ggacgcagtc tggggtcgaa gcttgtcggt tagggctgcc gtgacgtggt agagcagatg 1987 ggaccaagtg cggagctagg caggtgggtg gttgtggtgg tggcttacct tctgtaacgc 2047 aatggcatct catctcactc gcctgctccc tgattggtgg ctctgttcgg cctggcgctt 2107 tttgggaccg ctggctggaa tggattgctc cggaacgcca ggttgagctg ggctggcgcg 2167 agtagattgg ccgctccgag ctgcaaccat aataaaattt tcggaccctg taagccgcac 2227 ccgaccaggt ctccattggc ggacatgcac gacgtccttc gcaggcacgg cctgcccgcc 2287 tctgatcacc cgcagttcc gagtccgccc

<212> PRT

<213> Acremonium thermophilum <220> <221> misc_feature <222> (13) .. (13) <223> n means a, t, c, or g <400> 2

Met Arg Ser Ser Pro Phe Leu Arg Ala Ala Leu Ala Ala Ala Leu Pro: 1 5 10 15 • ·· • · • · * .1 1 Leu Ser Ala His Ala Leu Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys. 20 25 30 • · · * · · • 1:.::: Cys Lys Pro Ser Cys Gly Trp Pro Gly Lys Ala Ser val Asn Gin Pro:. ·. 35 40 45 • · 1 ··· · *** ·: ... 1 hr Phe Ser Cys Ser Ala Asp Trp Gin Arg lie Ser Asp Phe Asn Ala 50 55 60 • ·: .V Lys ser Gly Cys Asp Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gin Thr. ···. 65 70 75 80 * · ···

Pro Trp Ala val Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr Ala:.: 85 90 95 • · «· ···

Ile Ala Gly Gly Ser Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu: .1 100 105 110

Page 4 · 1 1 9325 2052060EN.ST25.txt

Thr Phe Asn ser Gly val Ala Gly Lys Thr Mat or val Gin Sen 115 120 125

Thr ser Thr Glv Gly Asp Leu Gly Ser Asn Gin Phe Asp Leu Ala lie 130 135 140 pro Glv Glv Glv val Gly lie Phe Asn Gly Cys Ala ser Gin Phe Gly 145 150 155 160

Glv Leu Pro Glv Ala Gin Tyr Gly Gly lie Ser Asp Arg ser Gin Cys y y 165 170 175

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

TrD Phe Gin Asn Area Asp Asn Pro Thr Phe Thr Phe Gin Arg Val 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 val Phe Asn Pro Pro Ser Gly Gly Ser Pro Ser Thr 225 230 235 240

Thr Ser Thr Thr Thr Ser Ser Pro Ser Gly Pro Thr Gly Asn Pro Pro 245 250 255

Gly Gly Gly Gly Cys Thr Ala Gin Lys Trp Ala Gin cys Gly Gly Thr 260 265 270

Gly Phe Thr Gly Cys Thr Thr Cys Val Ser Gly Thr Thr Cys Gin h ·. : 275 280 285 • · · • · • # · V * Gin Asn Gin Trp Tyr Ser Gin Cys Leu, 290 295 • · · · * #:. :: <2io> 3 • ·:.:: <2ii> 2033 · · ·

<212> DNA

<213> Acremonium thermophilum • · • · · · · · φ ··· Σ ...: <220> <22i> cds • · <222> (259) .. (702) • ··: · *. ·. <223> • · • · • ·

Page 5 <220> 2052060EN.ST25.txt 119325 <221> Intron <222> (703) .. (857) <223> <220> <221> CDS <222> (858) .. (888) <223> <220> <221> intron <222> (889) .. (990) <223> <220>

<221> CDS

<222> (991) .. (1268) <223> <400> 3 ctcgaggaga ggaaccgagt ttgaaagatg ctatatatcg atagactacc ggcgtcgcct 60 • · cgccctgtcc gctctcttgc attccccctg ttgatgagac gagacaaaat 120. aaaagatccg tcgccgagat ttcaccagtg gtaagtcccg agaattggtc attcgacgtt 180 ···:. *. caatatgagt gtcaaagcta tgggtcctaa caaagaagga agcaagagct ttaaagagac 240 ··· ·:. *. agaataacag cagcaaag atm cgt etc cca eta ccg act ctg etc gee etc 291 S ··: Met Arg Leu Pro Leu Pro Thr Leu Leu Ala Leu. * ··. 15 10 • · »·· ttg ccc tae tac etc gaa gtg tec get cag ggg gca tec gga ace ggc 339

Leu Pro Tyr Tyr Leu Glu val Ser Ala Gin Gly Ala Ser Gly Thr Gly 15 20 25 • · · • * aeg aca aca cgt tac tgg gat tgc tgc aag ccg age tgc geg tgg cct 387 * ··· * Thr Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys Ala Trp Pro 30 35 40 • · · • · t ctg aag ggc aat teg ccc age ccg gtg cag act tgc gac aag aat gac 435:: Leu Lys Gly Asn Ser pro Ser pro val Gin Thr cys Asp Lys Asn Asp T 45 50 55 · *: *: agg ccg ctg aac gat ggg gga aac acc aag tcc ggc tgc gac aac ggt 483 ...,:: Arg pro Leu Asn Asp Gly Gly Asn Thr Lys Ser Gly Cys Asp Asn Gly * * 60 65 70 75

Page 6 2052060EN.ST25.txt 119325 ggc gag gcc ttc recover tgc tea tee cag agt ccc tgg gee gtc aat gag 531

Gly Gly Ala Phe Met Cys Ser Ser Gin ser Pro Trp Ala val Asn Glu 80 85 90 ace ace age tae ggc tgg gca gcc gtt cgt ate gcc ggc agt ace gag 579

Thr Thr Ser Tyr Gly Trp Ala Ala Val Arg lie Ala Gly ser Thr Glu 95 100 105 teg gcc tgg tgc tgt gcc tgc tae gag etc acc ttc acc agt ggg ccc 627

Ser Ala Trp Cys Cys Ala cys Tyr Glu Leu Thr Phe Thr Ser Gly Pro 110 115 120 gtc agt gga aag aag etc ata gtc cag gcc aeg aac act ggt gga gac 675

Val Ser Gly Lys Lys Leu lie Val Gin Ala Thr Asn Thr Gly Gly Asp 125 130 135 ett ggg age aac cac ttt gac ett geg gtatgtgggg tttttctttc 722

Leu Gly Ser Asn His Phe Asp Leu Ala 140 145 ttcatcatcg ctctcaccat ggattcctcg gcgcaaggac caagattgag aagcgtcaat 782 gccgggttgg acacgggagc cgggatagga gcag gag gag gag gag 155 gtaggttcct tccctgaagt accggcaaca gcctgtgcgt tgctgtatac cccttttaat 948 catagcatct tcctgctgga tacaagccaa cccattttct ag ct tgc aeg aac 1001

Ala Cys Thr Asn 160 cag tat ggt geg ccc ccg aac ggc tgg ggc gac agg tat ggt ggc gtg 1049

Gin Tyr Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly Gly val 165 170 175 cac teg egg age gac tgc gac age ttc ccc geg geg etc aag gcc ggc 1097

His ser Arg Ser Asp Cys Asp Ser Phe pro Ala Ala Leu Lys Ala Gly 180 185 190 • · tgc tae tgg nor ttc gac tgg ttc cag ggc gcc gac aac ccg tee gtg 1145 • 'Cys Tyr Trp Arg Phe Asp Trp Phe Gin Gly Ala Asp Asn Pro Ser val:; ·; 195 200 205 210 • · · • · *. age ttc aaa cag gta gcc tgc ccg gca gcc ate aca get aag age ggc 1193 ...: ser Phe Lys Gin val Ala Cys Pro Ala Ala lie Thr Ala Lys Ser Gly • j '; 215 220 225 »··«. ***. tgt act ege cag aac gat gcc ate aac gag act ccg act ggg ccc age 1241 ··· Cys Thr Arg Gin Asn Asp Ala lie Asn Glu Thr Pro Thr Gly Pro Ser 230 235 240 · '· * · act gtg eet acc tae acc geg tea ggc tgaaagtcgg ctggggcacc 1288 • · Thr vaT Pro Thr Tyr Thr Ala Ser Gly: ** ·. 245,250 * ·· y. ' attgcccagg tgatggttgg gcatgtgtta gtctcactca ccagggacat ttgtcgcgac 1348 • · · ctgatcatag gcgccagggg agttgaaagg ggttgccgta egagaagaca ttttgtcgcc 1408 • · * · * gtcttactcc cagccacttc tgtacatatt caatgacatt acatagcccg caaatatgtt 1468 • «ϊ · · * catatatcgt ggccgcccaa accgccccgg tttgettagg ctggagctga agtggctcgc 1528 · · · · * cgatggctgt caaaggcagt eggaatatte ctcgttgctt cggcaacacg gtagctgctt 1588

Page 7 2052060FI.ST25.txt 119,325 gaaccgtacc cagcattaga acaccccccg ccgagggctt gctacgtcaa tggcggggtc 1648 tccaacccct gcgcggcaca aaaccaacca cgccctcgtc ttttatgatg tcctcgctca 1708 aacgtcccgt gacgacactc cgctcatggt ctggtcctct gatgtagaag gggtaggtca 1768 gccgatggtc gtcaccgtcg tcaatgcttc cctcaagctt cttgcggcct ttatcctcca 1828 actcttccca catgagaact ccatctttcc gccttttcac aaagccactg ccctccttgt 1888 caagggccaa aaaccaacgc cgctgatgaa tgcttccgat cgtgtttgac gcgcccgggg 1948 tatgcatttg gttcggcgca cttttttcgt cctccagctc ccttaactcc cgttccatct 2008 gagagggtga ctcgtctact cgact 2033 <210> 4 <211> 251

<212> PRT

<213> Acremonium thermophilum <400> 4

Met Arg Leu Pro Leu Pro Thr Leu Leu Area Leu Leu Pro Tyr Tyr Leu 15 10 15

Glu val Ser Ala Gin Gly Ala Ser Gly Thr Gly Thr Thr Thr Arg Tyr 20 25 30

Trp Asp Cys Cys Lys Pro ser Cys Ala Trp Pro Leu Lys Gly Asn Ser 35 40 45 • · V *: pro ser pro val Gin Thr Cys Asp Lys Asn Asp Arg Pro Leu Asn Asp 50 55 60 * · ·: .ϊ. : Gly Gly Asn Thr Lys Ser Gly Cys Asp Asn Gly Gly Gly Ala Phe Met:. ·. 65 70 75 80 • t · »·« · •:.:: Cys ser Ser Gin Ser Pro Trp Ala Val Asn Glu Thr Thr Ser Tyr Gly 85 90 95 • · ···

Trp Ala Ala val Arg lie Ala Gly ser Thr Glu Ser Ala Trp Cys Cys 100 105 HO

* · »• * • · ·

Ala cys Tyr Glu Leu Thr Phe Thr ser Gly Pro val ser Gly Lys Lys 115 120 i25 * I · • t * • ·: ***: Leu lie val Gin Ala Thr Asn Thr Gly Gly Asp Leu Gly Ser Asn His *; * 130 135 140 e · ·: * t:

Phe Asp Leu Ala lie pro Gly Gly Gly val Gly Gin Ser Asn Ala cys • · 145 150 155 lb0 page 8 2052060Fl.ST25.txt 119325

Thr Asn Gin Tyr Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly 165 170 175

Gly val His Ser Arg Ser Asp Cys Asp Ser Phe Pro Ala Ala Leu Lys 180 185 190

Ala Gly Cys Tyr Trp Arg Phe Asp Trp Phe Gin Gly Ala Asp Asn Pro 195 200 205

Ser Val Ser Phe Lys Gin Val Ala Cys pro Ala Ala lie Thr Ala Lys 210 215 220

Ser Gly Cys Thr Arg Gin Asn Asp Ala lie Asn Glu Thr Pro Thr Gly 225 230 235 240

Pro Ser Thr Val Pro Thr Tyr Thr Ala Ser Gly 245 250 <210> 5 <211> 17

<212> PRT

<213> Acremonium thermophilum <400> 5

Gin Ser Cys Ser Ser Phe Pro Ala pro Leu Lys Pro Gly Cys Gin Trp 15 10 15 **. : • · Arg • ♦ · ♦ · ·: · ί ·: <210> 6 • * * ·· ϊ <211> 13 • ·

ϊ ·· ί <212> PRT

· # «• · * ··· * <213> Acremonium thermophilum t»! * V <400> 6

• M

• «jyr Ala Leu Thr Phe Asn Ser Gly Pro val Ala Gly Lys 15 10 * · · • · • • <2io> 7: ·! ·. <2ΐι> ίο • «• · .... · <212> prt • ·

Page 9 2052060EN.ST25.txt 1 1 9325 <213> Acremonium thermophilum <400> 7

Val Gin Cys pro Ser Glu Leu Thr Ser Arg 1 5 10

<210> 8 <211> 13 <212> PRT

<213> Acremonium thermophilum <400> 8

Asn Gin pro val Phe Ser Cys Ser Ala Asp Trp Gin Arg 1 5 10 <210> 9 <211> 14

<212> PRT

<213> Acremonium thermophilum <400> 9

Tyr Trp Asp Cys Cys Lys pro Ser Cys Gly Trp Pro Gly Lys 15 10 • · Y *: <2ΐο> ίο «** V 1 <211> 4

<212> PRT

* · • * · ί ·! ! <213> Acremonium thermophilum * · • · · · · · · · · · · · · · · · · · · · · · · · · · · · ›

Pro Thr Phe Thr • · 1 * ψ · 1 • * · • · ··· <210> 11: Y: <2ii> 20 • · <212> dna • ♦ · <213> Artificial Sequence • * • · · * ··· • «

Page 10 <220> 2052060EN.ST25.txt 119325 <223> primer <220> <221> misc_feature <222> (3) .. (3) <223> y means c or t <220> <221> misc_feature <222 > (9) .. (9) <223> y means c or t <220> <221> misc_feature <222> (12) .. (12) <223> y means c or t <220> <221> misc_feature <222> (15) .. (15) <223> r stands for a or g • »• · · • · · · · ·:: <400> 11. . *. taytgggayt gytgyaarcc 20 · * · • ·: <2io> 12 • · · * .:: <2ii> 2i ··· • *

* · * · * <212> DNA

<213> Artificial Sequence • · * · · • · * • · • ·? -: <220>: V: <223> primer <220> • * * <221> mi sc_feature • · <222> (1) .. (1) ·

Page 11 119325 2052060EN.ST25.txt <223> r means a or g <220> <221> misc_feature <222> (4) .. (4) <223> r means a or g <220> <221> misc_feature < 222> ¢ 7) .. (7) <223> n means a, t, c or g <220> <221> misc_feature <222> (10) ..CIO) <223> r means a or g <220> <221> misc_feature <222> C13). -C13) <223> y means c or t • · ♦ · · · · · · M · • »« * · * * <220> • · · '· * · * <221> misc_feature • · Ϊ <222 > C16) .. C16) • · •> «··· · <223> r means a or g» ·· • * • * * · · <400> 12. *. * · Rttrtcngcr ttytgraacc a 21 * · · »«: ···: <210> 13 ϊ'ί'ϊ <211> 7 Ψ «

i "': <212> PRT

Ϊ *. ·. <213> Melanocarpus albomyces • · · page 12 2052060EN.ST25.txt 119325 <400> 13

Trp Phe Gin Asn Ala Asp Asn 1 5 <210> 14

<211> 636 <212> DNA

<213> Acremonium thermophilum <400> 14 tactgggatt gttgcaagcc gtcctgcggc tggccgggaa aggcctcggt gaaccagccc 60 gtcttctcgt gctcggccga ctggcagcgc atcagcgact tcaacgcgaa gtcgggctgc 120 gacggaggct ccgcctactc gtgcgccgac cagacgccct gggcggtcaa cgacaacttc 180 tcgtacggct tcgcagccac ggccatcgcc ggcggctccg agtccagctg gtgctgcgcc 240 tgctatgcgt gagttctctg caagccgctt cccacccccg ctttctgtgc aggccgcttc 300 ccccctaccc acccacttcc ccccccccgc ctctgtgatc gggcatccga gctaagttgc 360 gtgtcgtcca gactcacctt caactcgggc cccgtcgcgg gcaagaccat ggtggtgcag 420 tcgaccagca ccggcggcga cctgggcagc aaccagttcg acctcgccat ccccggcggc 480 ggcgtgggca tcttcaacgg ctgcgcctcc cagttcggcg gcctccccgg cgcccagtac 540 ggcggcatca gcgaccgcag ccagtgctcg tccttccccg cgccgctcca gccgggctgc 600 cagtggcgct tcgactggtt ccagaacgcg GATAAT 636 <210> 15 • · <211> 786 · · ·

: ·: J <212> DNA

: · Ί.Σ <213> Acremonium thermophilum • · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · 60 · 60 120 120aaaa gtggcggggc cttcatgtgc tcatcccaga gtccctgggc cgtcaatgag 180 • ·. ***. accaccagct acggctgggc agccgttcgt atcgccggca gtaccgagtc ggcctggtgc 240 ··· tgtgcctgct acgagctcac cttcaccagt gggcccgtca gtggaaagaa gctcatagtc 300 • • · · · · caggccacga acactggtgg agaccttggg agcaaccact ttgaccttgc ggtatgtggg 360 • · *** gtttttcttt cttcatcatc gctctcacca tggattcctc ggcgcaagga ccaagattga 420 • • · · · • · * * gaagcgtcaa tgccgggttg gacacgggag ccgggatagg aacacagagg ccgtttaaga 480 '* ccgtcagctg acagcaggag caattagatt cccggaggtg gtgttggtca gttcaatggt 540 119 325 Page 13 2052060FI.ST25.txt aggttccttc cctgaagtac cggcaacagc ctgtgcgttg ctgtataccc cttttaatca 600 tagcatcttc ctgctggata caagccaacc cattttctag cttgcacgaa ccagtatggt 660 gcgcccccga acggctgggg cgacaggtat ggtggcgtgc actcgcggag cgactgcgac 720 agcttccccg cggcgctcaa ggccggctgc tactggcgat tcgactggtt tcaaaacgcc 780 gacaac 786 <210> 16 <211> 994

<212> DNA

<213> Acremonium thermophilum <220> <221> CDS <222> (13) .. (95) <223> <220> <221> intron <222> (96) .. (154) <223> <220> • · ·

A: <221> CDS

<222> (155) .. (403) • · i ·:: <223> • · • · * • * · • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · : · ** ·: <222> (404) .. (526) • · *! ···: <22 3> • · * · · · · <220> ···

<221> CDS

• · <222> (527) .. (986) page 14 • · <223> 119325 2052060EN.ST25.txt <400> 16 ggactgcgca tc recover cgc tcc tea ccc ttt etc ege gca get ctg get gee 51

Met Arg Ser Ser Pro Phe Leu Arg Ala Ala Leu Ala Ala 15 10 get ctg cct ctg age gee cat gee etc gac gga aag teg time ag 95

Ala Leu Pro Leu Ser Ala His Ala Leu Asp Gly Lys Ser Thr Arg 15 20 25 gtatgccaat cctcgtacct ctgccctctg tagaaacaag tgaccgactg caaagacag 154 a tae tgg gac tgc tgc aag eeg tec tgc ggc tgg gee tegga

Tyr Trp Asp Cys Cys Lys Pro Ser cys Gly Trp Ala Gly Lys Ala Ser 30 35 40 gtg aac cag ccc gtc ttc teg tgc teg gee gac tgg cag cgc ate age 251 h Asn Gin Pro Val Phe Ser Cys Ser Ala Asp Trp Gin Arg lie ser 45 50 55 60 gac ttc aac geg aag teg ggc tgc gac gga ggc tec gee tae teg tgc 299

Asp Phe Asn Ala Lys Ser Gly cys Asp Gly Gly Ser Ala Tyr Ser Cys 65 70 75 gee gac cag aeg ccc tgg geg gtc aac gac aac ttc teg tae ggc ttc 347

Ala Asp Gin Thr Pro Trp Ala Val Asn Asp Asn Phe Ser Tyr Gly Phe 80 85 90 gca gee aeg gee ate gee ggc ggc tec gag tec age tgg tgc tgc gee 395

Ala Ala Thr Ala lie Ala Gly Gly Ser Glu Ser Ser Trp Cys Cys Ala 95 100 105 TGC TAT GC gtgagttctc tgcaagccgc ttcccacccc cgctttctgt 443 cys Tyr Ala 110 gcaggccgct tcccccctac ccacccactt cccccccccc gcctctgtga tcgggcatcc 503 gagetaagtt gcgtgtcgtc CAG a etc ace ttc aac teg ggc ccc gtc May 554 '·: Leu Thr Phe Asn Ser Gly Pro val Ala 115 120 • · ·; · *. ggc aag ace reverse gtg gtg cag teg acc age ace ggc ggc gac ctg ggc 602 '··· * Gly Lys Thr Met Val val Gin ser Thr ser Thr Gly Gly Asp Leu Gly • 125 130 135 • · · ·. *. age aac cag ttc gac etc gee ate ccc ggc ggc ggc gtg ggc ate ttc 650 ...: ser Asn Gin Phe Asp Leu Ala lie pro Gly Gly Gly Val Gly lie Phe 140 145 150 • · · aac ggc tgc gee tec cag ttc ggc ggc etc ccc ggc gee cag tae ggc 698

Asn Gly Cys Ala Ser Gin Phe Gly Gly Leu Pro Gly Ala Gin Tyr Gly 155 160 165 • ·: ***: 99C ate age gac cgc age cag tgc teg tec ttc ccc geg ccg etc cag 746 ··· Gly lie Ser Asp Arg Ser Gin cys Ser Ser Phe Pro Ala Pro Leu Gin 170 175 180 • * »• · * M ccg ggc tgc cag tgg cgc ttc gac tgg ttc cag aac gee gac aac ccc 794 ·.,. · Pro Gly Cys Gin Trp Arg Phe Asp Trp Phe Gin Asn Ala Asp Asn Pro. 185 190 195 200 * · · 1 *: · * acc ttc acc ttc cag cgc gtg cag tgc ccg tec gag etc aeg tec cgc 842 ....: Thr Phe Thr Phe Gin Arg val Gin Cys pro Ser Glu Leu Thr Ser Arg * * 205 210 215

Page 15 2052060EN.ST25.txt 119325 acg ggc tgt aag cgc gac gac gac gcc age tat ccc gtc ttc aac ccg 890

Thr Gly Cys Lys Arg Asp Asp Asp Asp Ala Ser Tyr Pro Val Phe Asn Pro 220 225 230 cct age ggt ggc tee ccc age ace acc age ace ace ace age tee ccg 938

Pro Ser Gly Gly Ser Pro Ser Thr Thr Ser Thr Thr Thr Ser Ser Pro 235 240 245 tee ggt ccc time ggc aac cct cct gga ggc ggt ggc tgc act gcc cag 986

Ser Gly pro Thr Gly Asn Pro Pro Gly Gly Gly Gly cys Thr Ala Gin 250 255 260 tgactgca 994 <210> 17 <211> 264

<212> PRT

<213> Acremonium thermophilum <400> 17

Met Arg Ser Ser Pro Phe Leu Arla Area Leu Area Area Area Leu Pro 15 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 hrs Phe ser cys ser Ala Asp Trp Gin Arg lie ser Asp Phe Asn Ala: 50 55 60 • * · • · * · «* · * * Lvs ser Gly cys Asp Gly Gly ser Ala Tyr ser Cys Ala Asp Gin Thr. 65 70 75 80 • · · • · ® ·:.:: Pro jrn Ala val Asn Asp Asn Phe Ser Tyr Gly Phe Ala Ala Thr Ala:. ·. 85 90 95 • · * # · t # ·

Tip Ala Gly Gly ser Glu Ser ser Trp Cys Cys Ala Cys Tyr Ala Leu 100 105 110 • * * .V Thr Phe Asn Ser Gly Pro Val Ala Gly Lys Thr Met val val Gin Ser. ···. 115 120 125 • · • * ·: Y: Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn Gin Phe Asp Leu Ala lie 130 135 140 • · • · ·· *: *! *. Pro Gly Gly Gly Val Gly lie Phe Asn Gly Cys Ala ser Gin Phe Gly:. * 145 150 155 l ° u

Page 16 • · 119325 2052060EN.ST25.txt

Gly Leu Pro Gly Alla Gin Tyr Gly Gly lie 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 val 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 Val Phe Asn pro Pro Ser Gly Gly Ser Pro Ser 240 225 230 235

Thr Ser Thr Thr Thr Ser Ser Pro Ser Gly Pr0 T ^ r G ^ y ASn 255 ΡΓ ° 245 250

Gly Gly Gly Gly Cys Thr Ala Gin 260 <210> 18 <211> 907

<212> DNA

<213> Acremonium thermophilum <220>

<221> CDS

• ♦ :: y <222> (13) .. (95) • · · '· *' <223> • · · • t I • · • ·: ·· ϊ <220> • · • · · ··· · · <221> intron * ··· * <222> (96) .. (154) <22 3> • »

I · I

• * I • · ·· »<220>

<221> CDS

O <222> (155) .. (403): v. <223> * · • · • ·

Page 17 <220> 2052060Fl.ST25.txt 119325 <221> Intron <222> (404) .. (526) <223> <220>

<221> CDS

<222> (527) .. (899) <223> <400> 18 ggactgcgca tc recover cgc tcc tea ccc ttt etc ege gca get ctg get gee 51

Met Arg Ser Ser Pro Phe Leu Arg Ala Leu Ala Ala 1 5 10 get ctg cct ctg age gee cat gee etc gac gga aag teg time ag 95

Ala Leu Pro Leu Ser Ala His Ala Leu Asp Gly Lys Ser Thr Arg 15 20 25 gtatgccaat cctcgtacct ctgccctctg tagaaacaag tgacegactg caaagacag 154 a tae tgg gac tgc tgc aag eeg tec tgc ggc tgg gee gga aga

Tyr Trp Asp Cys Cys Lys Pro Ser Cys Gly Trp Ala Gly Lys Ala Ser 30 35 40 gtg aac cag ccc gtc ttc teg tgc teg gee gac tgg cag ege ate age 251 h Asn Gin Pro Val Phe Ser Cys ser Ala Asp Trp Gin Arg lie Ser 45 50 55 60 gac ttc aac geg aag teg ggc tgc gac gga ggc tec gee tae teg tgc 299

Asp Phe Asn Ala Lys Ser Gly Cys Asp Gly Gly Ser Ala Tyr Ser Cys 65 70 75 • · · * · * · gee gac cag aeg ccc tgg geg gtc aac gac aac ttc teg tae ggc ttc 347 • Ala Asp Gin Thr Pro Trp Ala Val Asn Asp Asn Phe Ser Tyr Gly Phe: 80 85 90 * ·· • · ': gca gee aeg gee ate gee ggc ggc tec gag tec age tgg tgc tgc gee 395 ··· * Ala Ala Thr Ala lie Ala Gly Gly Ser Glu Ser Ser Trp Cys Cys Ala j95 loo 105 * ·· ·. *** «tgc tat gc gtgagttctc tgcaagccgc ttcccacccc cgctttctgt 443 ··· Cys Tyr Ala 110 • V. gcaggccgct tcccccctac ccacccactt cccccccccc gcctctgtga tcgggcatcc 503 • · · ***. gagetaagtt gcgtgtcgtc cag a etc acc ttc aac teg ggc ccc gtc May 554 ··· Leu Thr Phe Asn Ser Gly Pro val Ala, ..., 115 120 • · ·, ··· ggc aag acc recover gtg gtg cag teg acc age acc ggc ggc gac ctg ggc 602 * ... · Gly Lys Thr Met Val Val Gin Ser Thr Ser Thr Gly Gly Asp Leu Gly • 125 130 135 • · · • · · J · * age aac cag ttc gac etc gee ate ccc ggc ggc ggc gtg ggc ate ttc 650 ·. ·:: Ser Asn Gin Phe Asp Leu Alla Pro Pro Gly Gly Gly Val Gly lie Phe 140 145 150

Page 18 2052060EN.ST25.txt 119325 aac ggc tgc gcc tcc cag ttc ggc ggc etc ccc ggc gee cag tae ggc 698

Asn Gly Cys Ala Ser Gin Phe Gly Gly Leu Pro Gly Ala Gin Tyr Gly 155 160 165 ggc ate age gac ege age cag tgc teg tec ttc ccc geg ccg etc cag 746

Gly lie Ser Asp Arg Ser Gin Cys Ser ser Phe Pro Ala Pro Leu Gin 170 175 180 ccg ggc tgc cag tgg ege ttc gac tgg ttc cag aac gcc gac aac ccc 794

Pro Gly Cys Gin Trp Arg Phe Asp Trp Phe Gin Asn Ala Asp Asn Pro 185 190 195 200 acc ttc acc ttc cag ege gtg cag tgc ccg tec gag etc time tec ege 842

Thr Phe Thr Phe Gin Arg val Gin cys Pro Ser Glu Leu Thr Ser Arg 205 210 215 aeg ggc tgt aag ege gac gac gac gcc age tat ccc gtc ttc aac ccg 890

Thr Gly Cys Lys Arg Asp Asp Asp Asp Ala Ser Tyr Pro val Phe Asn Pro 220 225 230 cct age ggt tgactgca 907

Pro Ser Gly 235 <210> 19 <211> 235

<212> PRT

<213> Acremonium thermophilum <400> 19

Met Arg ser Ser Pro Phe Leu Arg Alla Leu Alla Alla Alla Leu Pro 15 10 15 • ·: · ** ·: Leu Ser Alla His Alla Leu Asp Gly Lys Ser Thr Arg Tyr Trp Asp Cys 20 25 30 • * · * "· * Cvs 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 lie Ser Asp Phe Asn Ala. ** \ 50 55 60 • · ♦ * ·

Lys Ser Gly Cys Asp Gly Gly ser Ala Tyr Ser Cys Ala Asp Gin Thr 65 70 75 80 • · • I * • f ***** Pro ttd Ala Val Asn Asp Asn Phe Ser Tyr Gly Phe Ala Thr Ala 85 90 95 • · · • * · • ·; '**: He Ala Gly Gly Ser Glu Ser Ser Trp Cys cys Ala Cys Tyr Ala Leu * "100 105 no ·· ·« * · • · ....: Thr Phe Asn Ser Gly Pro val Ala Gly Lys Thr Met val val Gin ser • · US 120 125

Page 19 2052060EN.ST25.txt 1 1 9325

Thr ser Thr Gly Gly Asp Leu Gly ser Asn Gin Phe Asp Leu Ala lie 130 135 140 pro Gly Gly Gly Val Gly lie Phe Asn Gly Cys Ala Ser Gin Phe Gly 145 150 155 160

Gly Leu Pro Gly Ala Gin Tyr Gly Gly lie 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 val 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 val Phe Asn Pro Pro Ser Gly 225 230 235 <210> 20 <211> 1175

<212> DNA

<213> Acremoni urn thermophilum <220> • · * 9 ·

• · <221> CDS

···! · ** 4: <222> CD (444) • · · ··· <223> • · • · I «* · · * · · * <220> ··· •« * ··· * <221> intron <222> (445) .. (599) • · · • t · • «<223> * ·· e · • I · ** * X! <220> · * ·

! ...: <221> CDS

• V. <222> (600) .. (630) • · ··· ♦ · <223>

Page 20 <220> 119325 2052060EN.ST25.txt <221> intron <222> (631) .. (732) <223> <220>

<22l> CDS

<222> (733) .. (1172) <223> <400> 20 atm cgt etc cca eta ccg act ctg etc gee etc ttg ccc tae tae etc 48

Met Arg Leu Pro Leu Pro Thr Leu Leu Area Leu Leu Pro Tyr Tyr Leu 15 10 15 gaa gtg tee get cag ggg gca tee gga ace ggc time aca aea cgt tae 96

Glu val Ser Ala Gin Gly Ala Ser Gly Thr Gly Thr Thr Thr Arg Tyr 20 25 30 tgg gat tgc tgc aag ccg age tgc geg tgg cct ctg aag ggc aat teg 144

Trp Asp Cys Cys Lys Pro Ser Cys Ala Trp Pro Leu Lys Gly Asn Ser 35 40 45 ccc age ccg gtg cag act tgc gac aag aat gac agg ccg ctg aac gat 192

Pro Ser Pro val Gin Thr Cys Asp Lys Asn Asp Arg Pro Leu Asn Asp 50 55 60 ggg gga aac acc aag tee ggc tgc gac aac ggt ggc ggg gee ttc atm 240

Gly Gly Asn Thr Lys Ser Gly cys Asp Asn Gly Gly Gly Ala Phe Met 65 70 75 80 • · · *** · tgc tea tee cag agt ccc tgg gee gtc aat gag acc acc age tae ggc 288 * Cys Ser Ser Gin Ser Pro Trp Ala Val Asn Glu Thr Thr ser Tyr Gly; ; *: 85 90 95 · * · Ϊ tgg gca gee gtt cgt ate gee ggc agt acc gag teg gee tgg tgc tgt 336 * ·· 'Trp Ala Ala Val Arg lie Ala Gly Ser Thr Glu Ser Ala Trp Cys Cys:: * j 100 105 no ·· # · · '** · gee tgc tae gag etc acc ttc acc agt ggg ccc gtc agt gga aag aag 384 ··· Ala Cys Tyr Glu Leu Thr Phe Thr Ser Gly Pro Val ser Gly Lys Lys 115 120 125 etc ata gtc cag gee aeg aac act ggt gga gac ett ggg age aac cac 432 * · Leu lie val Gin Ala Thr Asn Thr Gly Gly Asp Leu Gly Ser Asn His:: 130 135 140 * a ·, «* ·. ttt gac ett geg gtatgtgggg tttttctttc ttcatcatcg ctctcaccat 484 V, · Phe Asp Leu Ala. 145 • ♦ ggattcctcg gcgcaaggac caagattgag aagcgtcaat gccgggttgg acacgggagc 544 • · · * \ cgggatagga acacagaggc cgtttaagac cgtcagctga cagcagagca attag att 602 ··. «: Lie

Page 21 2052060EN.ST25.txt

1 1 9 λ 9 R

ccc gga ggt ggt gtt ggt cag tee aat g gtaggttcct tccctgaagt 650

Pro Gly Gly Gly or Gly Gin Ser Asn 150 155 accggcaaca gcctgtgcgt tgctgtatac cccttttaat catagcatct tcctgctgga 710 tacaagccaa cccattttct ag et tgc acg aac cag tat ggt gcg ccc eeg 761

Ala Cys Thr Asn Gin Tyr Gly Ala Pro Pro 160 165 aac ggc tgg ggc gae agg tat ggt ggc gtg cac teg egg age gae tgc 809

Asn Gly Trp Gly Asp Arg Tyr Gly Gly or Hi s Ser Arg Ser Asp Cys 170 175 180 gae age ttc ccc gcg gcg etc aag gee ggc tgc tae tgg cga ttc gae 857

Asp Ser Phe Pro Ala Ala Leu Lys Ala Gly Cys Tyr Trp Arg Phe Asp 185 190 195 200 tgg ttc cag ggc gee gae aac eeg tee gtg age ttc aaa cag gta gee 905

Trp Phe Gin Gly Ala Asp Asn Pro Ser Or Ser Phe Lys Gin Or Ala 205 210 215 tgc eeg gca gee ate aca get aag age ggc tgt act cgc cag aac gat 953

Cys Pro Ala Ala Ile Thr Ala Lys Ser Gly Cys Thr Arg Gin Asn Asp 220 225 230 gee ate aac gag act eeg act ggg ccc age ggt ggc tee ccc age acc 1001

Ala Ile Asn Glu Thr Pro Thr Gly Pro Ser Gly Gly Ser Pro Ser Thr 235 240 245 acc age acc acc acc age tee eeg tee ggt ccc acg ggc aac eet eet 1049

Thr ser Thr Thr Thr Ser Ser Pro Ser Gly Pro Thr Gly Asn Pro Pro 250 255 260 gga ggc ggt ggc tgc act gee cag aag tgg gee cag tgc ggc ggc act 1097

Gly Gly Gly Gly Gly Cys Thr Ala Gin Lys Trp Ala Gin Cys Gly Gly Thr 265 270 275 280 ggc ttc acg ggc tgc acc acc tgc gtc tcg ggc acc acc tgc cag gtg 1145 ,. Gly Phe Thr Gly Cys Thr Thr Cys Vai Ser Gly Thr Thr cys Gin Vai: ·: 285 290 295 • · cag aac cag tgg tat tee cag tgt ctg tga 1175 * Gin Asn Gin Trp Tyr ser Gin cys Leu! ; : 300 305 f * · e · • »t:, i: <210> 21 • · * * ·! ;; t! <2ii> 305 • · ** ·· * <212> prt. , <213> Aeremonium thermophilum • e »• i« • · · * · • * *: ** <400> 21 • ', V Met Arg Leu pro Leu Pro Thr Leu Leu Alla Leu Pro Tyr Tyr Leu. · * ·. 1 5 10 15 • · · «· j * · * · Glu or ser Ala Gin Gly Ala ser Gly Thr Thr Thr Thr Aro Tvr * *, 20 25 30 y y

• I

Page 22 119325 2052060EN.ST25.txt

Trp Asp Cys Cys Lys Pro Ser cys Lower Trp Pro Leu Lys Gly Asn Ser 35 40 45 pro Ser Pro val Gin Thr cys Asp Lys Asn Asp Arg Pro Leu Asn Asp 50 55 60

Gly Gly Asn Thr Lys ser Gly cys Asp Asn Gly Gly Gly Ala Phe Met 65 70 75 80

Cys Ser Ser Gin Ser Pro Trp Ala val Asn Glu Thr Thr Ser Tyr Gly 85 90 95

Trp Ala Ala Val Arg lie Ala Gly Ser Thr Glu Ser Ala Trp cys Cys 100 105 110

Ala Cys Tyr Glu Leu Thr Phe Thr Ser Gly Pro Val Ser Gly Lys Lys 115 120 125

Leu lie Val Gin Ala Thr Asn Thr Gly Gly Asp Leu Gly Ser Asn His 130 135 140 Phe Asp Leu Ala lie Pro Gly Gly Gly val Gly 01 ° Ser Asn Ala Cys 145 150 155 160

Thr Asn Gin Tyr Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly 165 170 175

Gly val His Ser Arg ser Asp Cys Asp Ser Phe Pro Ala Ala Leu Lys 180 185 190

Ala Gly Cys Tyr Trp Arg Phe Asp Trp Phe Gin Gly Ala Asp Asn Pro. ·. : 195 200 205 • ·· • · '· * * ser val ser Phe Lys Gin val Ala cys Pro Ala Ala lie Thr Ala Lys. 210 215 220 • · · · «· •:.: Ϊ ser Gly cys Thr Arg Gin Asn Asp Ala lie Asn Glu Thr Pro Thr Gly: 225 230 235 •« · «·· * * ··· * pro Ser Gly Gly Ser Pro Ser Thr Thr Ser Thr Thr Ser Ser Pro 245 250: .V ser Gly Pro Thr Gly Asn Pro Pro Gly Gly Gly Gly Cys Thr Ala Gin

···. 260 265 4 / U

• * ···: Yi lvs Trp Ala Gin Cys Gly Gly Thr Gly Phe Thr Gly Cys Thr Thr Cys * * y 275 280 • · • · ♦ · * ·. val Ser Gly Thr Thr cys Gin Val Gin Asn Gin Trp Tyr Ser Gin cys Ϊ. * 290 295 «·

Page 23 119325 2052060EN.ST25.txt

Leu 305 • · • · «« ft · ft · »··« ft · ft ft · ft «• 1 · • · • • ft • • • ft · · ft. «(··· · • • ft« 1 ft · ft ·· »· ft ft 1 • ft 1 ft ft • ft · ft · ft · • ft ft • ft · ft · · ft ft page 24 ft1 • · ft · • • ft ft • ft ft ϊ ·: • ·

Claims (21)

An endoglucanase polypeptide, characterized in that it contains a fragment having cellulolytic activity and selected from the group consisting of: (a) a polypeptide containing an amino acid sequence which is at least 80% identical to sequence # 2; and (b) a fragment of a) having cellulolytic activity. 2. Endoglucanase polypeptide according to claim 1, characterized in that its amino acid sequence is at least 85%, preferably 90%, more advantageous 95%, and most advantageously 98% identical to sequence # 2. Endoglucanase polypeptide according to claim 1, characterized in that it has an amino acid sequence according to sequence # 2. The endoglucanase polypeptide of claim 1, characterized in that the fragment has an amino acid sequence of sequence # 17 or sequence # 19. 5. Endoglucanase polypeptide according to claim 1, characterized in that it can be obtained from or derived from Acremonium sp., Preferably Acremonium thermophilum. The endoglucanase polypeptide of claim 5, characterized in that Acremonium sp. is CBS 116240. * · * ί Γ An isolated polynucleotide, characterized in that it encodes an endoglucanase polypeptide according to any one of claims 1 to 6. An isolated polynucleotide according to claim 7, characterized in that it has a nucleotide sequence selected from the group consisting of: • · · (a) a sequence according to sequence # 1, sequence # 16 or sequence No. 18, (b) a complementary strand of (a): V: (c) a fragment containing at least 20 nucleotides of (a) or (b); ***: 30 and (d) a sequence which is degenerate as a result of the genetic code in relation to (a), (b) or (c) defined sequence. The expression vector, characterized in that it contains a: ***: polynucleotide sequence encoding an endoglucanase polypeptide according to any of the claims: ··· 35 claims 1 - 6. A host cell, characterized in that it contains an expression vector according to claim 9. Host cell according to claim 10, characterized in that it is derived from a fungus. 5 Host cell according to claim 11, characterized in that it is Trichoderma reesei. A method for producing an endoglucanase polypeptide according to the claims patent claims 1-6, characterized by the abatement process comprising a step of growing a host cell according to any of claims 10-12. Enzyme preparation, characterized in that it contains a doglucanase polypeptide according to any one of claims 1-6. A method of biostate washing, characterized by the method comprising a step of adding an endoglucanase polypeptide according to any one of claims 1-6, or a preparation according to claim 14 to a fabric or garment containing cotton, such as denim. A method for biofinishing, characterized by the process comprising a step of adding an endoglucanase polypeptide according to any of claims 1-6, or a preparation according to claim 14 to a textile material such as fabrics, clothing or trees. A detergent composition, characterized in that it contains an endoglucanase polypeptide according to any one of claims 1-6, or a preparation according to claim 14 and auxiliaries, such as surfactants, surfactants, bleaches or fillers. . A method for treating textile material containing cellulose safer, characterized in that in the method, the textile material is brought into contact with a detergent composition according to claim 17. A method of treating a wood-derived pulp or fiber, characterized in that the method comprises a step of adding a doglucanase polypeptide according to any of claims 1-6, or a preparation according to claim 1. Claim 14 in a wood-derived or mechanical pulp or secondary fiber. ·· " 20. A method for improving the quality of animal feed, characterized in that the plant material is treated with an endoglucose in the process. nasal polypeptide according to any of claims 1-6 or with a preparation according to claim 14. ·· · ϊ V 35 21. Escherichia co // - strain with deposit number DSM 17324. * • ·