Cellulase fusion proteins and use thereof

FIELD: chemistry.

SUBSTANCE: fused proteins contain an endoglucanase nucleus amino acid sequence having at least 95% identity to SEQ ID NO:2, fused with an amino acid sequence containing a linker and a cellulose-binding domain (CBD), having at least 95% identity to SEQ ID NO:15. Such fused proteins can be obtained via a recombinant technique using suitable polynucleotides, expression vectors and host cells.

EFFECT: invention provides cellulase, having low activity with respect to restaining, and can be used to treat cellulose material; disclosed fused proteins and enzyme preparations based thereon can be used to prepare detergent compositions or for improving quality of animal feed.

26 cl, 8 dwg, 10 tbl, 10 ex

 

The SCOPE of the INVENTION

The invention relates to enzyme technology and, more precisely, to cellulase proteins merge that contains a catalytic domain and a cellulose-binding domain. Proteins merge can be obtained using recombinant technology using polynucleotides expressing vectors and host cells, which are also included in the scope of the invention. Proteins mergers and enzyme preparations on their basis is useful for the treatment of cellulosic material, for example textile material. In addition, fusion proteins can be used to obtain pulp and paper industry, for the extraction of oils from plants, preparation of detergent compositions or to improve the quality of animal feed. The invention also relates to a method for processing cellulosic material fusion proteins, in particular, to a method of biological abrasive machining (biostoning) or final biological trim (biofinishing) fabrics or clothing, especially denim. The invention also relates to detergent compositions and for animal feed containing proteins merge,

The LEVEL of TECHNOLOGY

Cellulose is the main structural component of higher plants and exist in virtually purified form only in cotton. It gives rise is entrusted cells high tensile strength, provide resistance to mechanical stress and osmotic pressure. Cellulose is a linear polysaccharide of glucose residues connected by β-1,4-bonds. In nature, cellulose is usually associated with lignin and hemicellulose. Cellulosic material decomposes in nature under the influence of various organisms, including bacteria and fungi. Biological conversion of cellulose into glucose is usually carried out three major groups of enzymes: cellobiohydrolase (VSS), endoglucanase (EG) and beta-glucosidase (BG).

Cellulase have a wide industrial application. In the textile industry cellulase is used for final finishing of denim to make clothing from denim fashionable shabby appearance, which is usually provided in the wash denim with pumice stones. In addition, cellulase are used, for example, for the elimination of musictaste tissues and prevent the formation of nodules on the surface of cotton clothing. As detergent cellulase are used to lighten the color and prevent Pomerania and conversion service. Cellulase is also used in the food industry and in the production of feed for animals and have a great potential in the production of pulp and paper industry, for example, to remove the ink from the fiber surfaces and improved and drainage of pulp.

Industrial used cellulase often represent a mixture of enzymes with different activity and substrate specificity. Commercial enzyme preparations often have activity all three cellulases: VSS, EG and BG. In addition, the unique properties of each cellulase is more suited to some purposes than others, and therefore have been some attempts to create and use of cellulases having only the desired activity. The most widely used cellulase is obtained from the fungi Trichoderma reesei. However, to obtain cellulases can be used and other fungi (see, for example, US 5457046).

The cellulase used for treatment of denim, usually divided into two main groups: acidic and neutral. Acid cellulase usually operate at pH 4, 0 to 5, 5 and the neutral cellulase operate at pH 6 to 8. Cellulase having the properties of both acidic and neutral cellulases can be called a hybrid cellulases. Acid cellulase used mainly for biostoning, derived from Trichoderma reesei (sexual form of Hypocrea jecorina), and neutral cellulase derived from a variety of fungi, including Melanocarpus, Myceliophthora, Fusarium, Acremonium, and Chrysosporium (Haakana et al. 2004). Enzymes derived from T. reesei, include, for example, cellulase family glycosylglycerols 5 (endoglucanase II, EG II)family 7 (cellobiohydrolase I CBHI) and family 12(endoglucanase III, EGIII; Ward et al. 1992), and neutral cellulase, mostly endoglucanase family 45 and family 7 (Henrissat, 1991; Henrissat and Bairoch, 1993, 1996).

In the patent US 5874293 disclosed improved cellulase composition containing increased amounts of endoglucanase EG II from T. reesei, for processing containing pulp tissue. The composition improves the color properties of tissues, increases their ease of use and appearance, and reduces the formation of nodules on the surface of the tissue. In the application WO 97/14804 described cellulase mol. mass of 20 kDa and 50 kDa with endoglucanase activity obtained from Melanocarpus sp., which is particularly effective in the textile industry and in the manufacture of detergents. It is assumed that to create a new enzymatic properties will be used fusion proteins containing cellulase mol. mass of 20 kDa and 50 kDa, combined with a cellulose-binding domain derived from Trichoderma reesi. However, in WO 97/14804 not given any specific examples of the preparation of such proteins and not their properties are described.

There is still a need for cellulases with improved properties, including endoglucanase that are most effective for the treatment of tissue, and for use in areas where commonly used cellulase. In particular, there is a continuing need for more efficient cellulases to improve the economic process is economical. The present invention is to meet these needs.

In the textile industry in recent years, much interest from manufacturers of denim causes the technology to create the appearance of tissue called "washing with pumice stones" (stone washing) or "digestion" of the fabric. Traditional washing with pumice stones reduces the strength of the fabric and increases the load on the washing machine. In the final processing of denim increasingly used enzymes and cellulase replace pumice stones or used in conjunction with them to make the desired tissues "worn" look. Controlled processing enzymes results in less damage to clothing and machinery and eliminates the use of stones.

The main problem associated with the use of enzyme abrasive washing, is to restore the color of the fabric due to the redistribution remote Indigo dye during or after biostoning. Redistribution of Indigo dye reduces the required contrast between the white and colored threads that can be more easily seen on the reverse side of the denim and the inner surface pockets (large blue). On the front side it may look like a reduced contrast between the colour and the areas from which the dye was removed in% the SSE of biostoning. Restoration of color can be reduced by using agents that hinder this recovery, for example, nonionic ethoxylated alcohols during tissue processing or the addition of bleaching agents on the stages of opolaskivanija. The nature of the enzyme affects the recovery of the painting. In General, neutral cellulase help restore color to a lesser extent than acidic cellulase.

In the application WO 97/09410 indicated that adding a certain type of cellulase to other cellulases, contributing "abrasion" tissue, reduces the recovery of the painting. Additional cellulase refers to a family of 5 or 7, but in itself has no significant "abrasive" activity. Preferably, the specified additional cellulase is from Bacillus or Clostridium.

In the patent US 5916799 described cellulase composition containing cellobiohydrolase and endoglucanase, which are used to limit proteolysis due to the separation of the nucleus and binding domains of the enzymes. The obtained enzyme composition was found to reduce the restoration of the painting. In the application WO 94/07983 noted that the redistribution of the dye in the tissue during the process of biostoning can be reduced using the composition of cellulases from fungi, which is essentially free from cellobiohydrolase komponentov application WO 96/23928 described treatment of tissue, containing cellulose, truncated cellulases. Such truncated enzymes that do not have cellulose-binding domain (CBD)was found to reduce the redistribution of the dye and increase the "abrasion".

The main conclusion that can be drawn when analyzing the above reference material, is that the cellulose-binding domain contributes to the restoration of the painting. So, the present invention provides a cellulase having low activity in relation to the recovery of the painting, despite the presence of cellulose-binding domain.

BRIEF description of the INVENTION

The present invention is based on the unexpected discovery of the fact that the effect endoglucanase can be considerably enhanced when connecting it with certain cellulose-binding domain without affecting the activity in relation to the recovery of the painting.

The invention relates to new proteins merge endoglucanase with improved hydralazine properties, for use in the textile industry, especially in the production of denim with biostoning. The claimed fusion proteins have the advantage that they are active both at acidic and neutral pH values, and therefore more effective when used in order to beallitani. When processing denim fusion proteins provide a low effect of the reduction is the service of painting. The use of more efficient endoglucanases in accordance with the invention is economical. Additional advantages are that facilitates transportation and storage of enzyme products, as they are used in smaller quantities. Proteins merger is also useful for use in detergent compositions and in other areas, for example, in the food industry, the extraction of oils from plants or in the production of pulp and paper.

The object of the invention are also polynucleotide encoding new proteins merge endoglucanase.

The object of the invention is also a method of obtaining proteins merge.

Another object of the invention relates to a new expressing vectors containing the specified polynucleotide, useful for protein merge endoglucanase, and new owners transformed such expressing vectors.

Another object of the invention relates to enzyme compositions containing one or more proteins merge endoglucanase.

The object of the invention are also methods of processing cellulosic material fusion protein, for example, for use in the textile industry, for the preparation of detergent compositions, animal feed, for the extraction of oils from plants, for the production of cellulosome and paper and in particular, for final processing of fabrics, especially for biostoning and biofinishing denim.

Another object of the invention relates to a feed for animals and detergent compositions containing proteins of the merger.

The invention also relates to cellulase the fusion protein containing the first amino acid sequence endoglucanase core and a second amino acid sequence containing the linker and cellulose-binding domain (CBD)that has at least 75%identity with SEQ ID NO:15, or a variant or fragment of the specified protein with cellulose-binding activity.

The invention also relates to the selected polynucleotide selected from the group including:

a) the nucleotide sequence of SEQ ID NO:3 or 5, or a sequence encoding a fusion protein of the cellulase according to claim 1;

b) a sequence complementary specified in (a);

C) the sequence obtained in accordance with the degeneracy of the genetic code, based on the sequences of a) and b).

The invention also relates to expressing the vector containing the specified nucleotide sequence, and the cell host containing expressing vector, and enzyme preparations containing protein merge.

The invention also relates to a method for producing a fusion protein, including Osamu transformation of a host cell expressing vector, encoding this protein fusions, and the cultivation of the specified host cell under conditions enabling expression of the indicated protein fusions, and, if necessary, separation and purification of the protein of the merge.

The invention also relates to a method of processing cellulosic material, providing contacting cellulosic material with a protein of the merger.

The invention also relates to methods of biological abrasive tissues, including the state security contact cellulase fusion protein or enzyme preparation with fabric denim or clothing from denim, and how the final biological treatment of tissues, including the state security contact cellulase fusion protein or enzyme preparation with textile materials such as cloth, or clothing, or thread.

The invention also relates to detergent compositions containing the fusion protein and detergent additives, feed for animals containing protein fusions, and the Escherichia coli strain deposited under number E. coli DSM 18159.

Specific embodiments of the invention are reflected in the claims.

Other objects, details and advantages of the invention will become apparent from the presented figures, detailed description, and examples.

BRIEF DESCRIPTION of DRAWINGS

Figure 1 shows a diagram of the expression cassettes used to transform protoplasts of Trichoderma reesi to get cellulase EG28 or EG28+CtCBD Thermoascus aurantiacus. Cel5A gene or cel5A_Ctcel7A linker CBD is under the control of the cbhI promoter Trichoderma reesi (cbhI prom) and termination of transcription is achieved by adding terminator (cbhI term). In the cartridge is built amdS gene (amdS) for selection of transformants. Expression cassette for receiving EG28 and EG28+CICBD isolated in the form of a NotI fragment size of 8.6 kb of pALK1930 or NotI fragment size of 8.9 kb of pALK1948, respectively.

Figure 2 shows the dependence of the activity heterologic produced cellulase EG28 T. aurantiacus selected from the culture supernatant, the pH when using CMC as a substrate for 10 min at 150°C (A). Temperature optimum cellulase EG28 determined at the optimal pH (6,0). The reaction mixture containing CMC as substrate, test for 10 min (B). Temperature optimum and the optimum pH fused protein EG28+CtCBD coincide with those of cellulase EG28 wild-type.

Figure 3 shows the effect of blasting the cellulase EG28 determined by measuring the color intensity, at different temperatures and pH 6.

Figure 4 shows the effect of blasting the cellulase EG28+CtCBD determined by measuring the color intensity, at different pH and at a temperature of 50 the C.

Figure 5 shows the effect of blasting the cellulase EG28+CtCBD determined by measuring the color intensity, at different pH and temperature of 60°C.

Figure 6 shows the effect of blasting the cellulase EG28+CtCBD determined by measuring the color intensity, at different temperatures and pH 6.

7 shows the effect of temperature granulation on the beta glucanase activity defined in the stern, which was added cellulase EG28.

On Fig the temperature effect of granulation on the beta glucanase activity defined in the stern, which was added cellulase EG28+CtCBD.

DETAILED description of the INVENTION

Cellulose containing catalytic domain/kernel (CD) to detect cellulase activity. In addition, the catalytic domain of the molecule in the cellulase may include one or more cellulose-binding domains (CBDs), also called "uglevodsvyazyvayushchie domains/modules" (CBD/CBM), which can be localized at the N - or C-terminal parts of the catalytic domain CBDs have uglevodsvyazyvayushchie activity and is able to mediate the binding of cellulase with crystalline cellulose, but do not possess hydrolytic activity, or have a very low hydrolytic activity on soluble substrates. Two of the above domain is usually connected through the flexible and highly glycosylated linker section, here called the linker. Such constructions are described, for example, Srisodsuk et al., 1993. Some of the natural endoglucanases and cellobiohydrolases have a cellulose-binding domain (CBD), while others in this domain do not have.

Endoglucanases (EGs) are one of three types of cellulases, usually required for biological degradation of cellulose to glucose. Endoglucanase break down internal beta-1,4-glucoside chain, while cellobiohydrolase otscheplaut disaccharide to cellobiose from the end of the cellulose polymer chain, and beta-1,4-glucosidase hydrolyzing cellobiose and other short calorigenically to glucose. The endoglucanases (EG) in accordance with the present invention is classified as AS. It is a beta-D-glucan-4-glucanohydrolase and catalyzes endogenous 1,4-beta-D-glycosidic linkages in the glucose polymers such as cellulose. Some endoglucanase able to hydrolyze, for example, 1,4-linkages in beta-D-glucan, also containing 1,3-linkages. They can also be classified as endo-1,3(4)-beta-glucanase (EC). Thus, the enzyme can catalyze the reaction on different substrates and belong to different classes.

Cellulase, including endoglucanase can also be classified according to their belonging to different families of glycosylglycerols in accordance with the PE the primary sequence, that is confirmed by the analysis of three-dimensional structures of some family members (Henrissat 1991, Henrissat and Bairoch, 1993, 1996). For example, the family 45 (previously celK) contains endoglucanase (EC) and a family of 5 (previously celA) contains mainly endoglucanase (EC). Family 7 (formerly family of cellulases celC) contains as endoglucanase and cellobiohydrolase. Some glycosylglycerols are multifunctional enzymes containing a catalytic domains belonging to different families of glycosylglycerols. For the purposes of the present invention endoglucanase part of the fusion protein, preferably belongs glycosylceramide family 45 or family of 5 and, more preferably, to a family of 5.

In accordance with the present invention endoglucanase part of the fusion protein obtained from fungi, preferably of the genus Thermoascus, and, more preferably, Thermoascus aurantiacus. This endoglucanases are described, for example, Hong et al., 2003. In the application WO 03/062409 assume the use of this enzyme for food, because in addition endoglucanases it also has beta-glucanase activity. More preferably, the endoglucanases derived from strain ALKO4242 Thermoascus aurantiacus deposited with the CBS under the number 116239. Endoglucanases specified gene of strain built into the plasmid pALK1926, which is deposited under the number DSM 17326. The protein encoded on the authorized genome labeled as "The EG28" or simply "EG28".

Alternatively, endoglucanase part of the fusion protein can be obtained from Acremonium sp. preferably, the A. thermophilum, and, more preferably, from strain ALKO4245, deposited under number CBS 116240. The endoglucanases derived from this strain and the encoded gene cel45A, designated as "At EG40" or simply "EG40".

Used herein, the term "endoglucanase core" means catalytic domain/kernel (CD) enzyme having at least endoglucanase activity. Such catalytic domain can be represented natural (native) form, or may be modified.

In accordance with one embodiment of the invention relates to endoglucanase core having at least, 75, 80, 85, 90, 95, 98 or 99% identity with SEQ ID NO:2 (The EG28). Preferably, the core contains at least the Mature protein, which corresponds to amino acids 19 to 334 of SEQ ID NO:2. The signal sequence was predicted using the program SignalP V3.0 (Nielsen et al., 1997; Bendtsen et al., 2004); the value of the NN obtained using neural networks and value MMOs when using hidden Markov models. Alternatively, endoglucanase part of the fusion protein has at least, 75, 80, 85, 90, 95, 98 or 99%identity with endoglucanase core At EG40 encoded by polynucleotides containing SEQ ID NO:8. Prefer the Ino, the core contains at least the Mature protein, which corresponds to amino acids 22 to 297 EG40.

Used herein, the term "cellobiohydrolase" or "VSS" refers to enzymes that otscheplaut pulp from the end of the glucose chain and produce cellobiose. They are also called 1,4-beta-D-glucan by cellobiohydrolase or cellulose 1,4-beta-cellobioside. These enzymes hydrolyzing 1,4-beta-D-glucoside communication pampering or non ends of the polymer containing such communication, for example, cellulose, with the formation of cellobiose.

CBD, including the linker, preferably obtained from Chaetomium thermophilum and, in particular, from cellobiohydrolase (CBHI/Cel7A), encoded by the genome of strain ALKO4265, deposited under number CBS 730.95. This CBD, including the linker, referred to as CtCBD". According to a preferred variant of the invention, the linker is connected to the cellulose-binding domain has a sequence at least 80, 85, 90, 95, 98, or 99% identical to SEQ ID NO:15 (which corresponds to amino acids 335-415 SEQ ID NO:4). According to another preferred variant of the invention, the second amino acid sequence contains the amino acids 335-379 SEQ ID NO:4.

The term "originating" in relation to the organism means that the polypeptide can be naturally produced MC is related to the specific microorganism or polynucleotide, encoding the polypeptide can be isolated from this organism. This term also refers to the cell host, which has a built-polynucleotide from the specified microorganism encoding the polypeptide. However, not excluded minor modification sequence, for example, substitutions, divisions, insertion and/or inversion of several amino acids/codons so that there is biological activity of the encoded polypeptide.

The core and linker + CBD, respectively, may be a fragment or variant of these two sequences, with the specified fragment or variant have cellulase activity and/or cellulose-binding activity. For example, the first amino acid sequence may contain a fragment or variant amino acid sequence having at least 75%identity with SEQ ID NO:2 or 8, and a second amino acid sequence may contain a fragment or variant amino acid sequence having at least 75%identity with SEQ ID NO:15.

Used herein, the term "cellulase activity" means catalytic ability to hydrolyze cellulose or its derivatives, for example, endoglucanase or beta-glucanase activity. In addition, endoglucanase and/or beta-glucanase activity of some cellulases can stage lunatica hemicellulases and/or xylanase activity.

Used herein, the term "identity" refers to the global identity between the amino acid sequences when compared with each other, starting with the first amino acid encoded by the corresponding gene to the last amino acid. The full identity of the sequences was measured using a global alignment Needleman-Wunsch while ensuring EMBOSS (European Molecular Biology Open Software Suite; Rice et al., 2000), version 3.0.0 with the following parameters: EMBLOSUM62, the free-space 10.0, fine length 0.5. The algorithm described in Needleman-Wunsch (1970). Specialist it is clear that the results obtained using the algorithm of Needleman-Wunsch, are comparable only when the alignment of the respective domains of the sequence. Thus, comparison of, for example, cellulase sequences, including CBD or signal sequences, sequences that do not contain these elements, is not valid.

In accordance with one embodiment of the invention, the fusion protein contains endoglucanases nucleus encoded by the genome, equivalent gene, integrated in E. coli DSM 17326. Preferably, the fusion protein is encoded merged genome equivalent gene, integrated in E. coli DSM 18159. "Equivalence" means here is practical a likeness or similarity. In accordance with the specification of the specific variant of the invention, the fusion protein contains endoglucanases the core, including the sequence of SEQ ID NO:2 and the linker and CBD, including SEQ ID NO:15. In particular, the core and linker + CBD include the sequence of SEQ ID NO:4 or 6, or a variant or fragment of these sequences with cellulase and cellulose-binding activity.

Used herein, the term "fragment" refers to part of a specific amino acid sequence that has a length sufficient to maintain a given biological activity. In other words, the fragment can be, for example, only the Mature part of the amino acid sequence or even a fragment of the Mature part. Variant specific amino acid sequence refers to an amino acid sequence that is not identical to the specific amino acid sequence and contains at least some amino acid changes, i.e., the deletion, substitution, inversion, insertion, etc. that do not have a significant impact on the biological activity of the protein compared with the activity of specific amino acid sequences for use in the desired order. Biological activity in the context of the present invention relates to cellulase activity, ability to bind cellulose or both of these activities.

Protein fusions, according to the claimed invention, can be half the ene by attaching endoglucanases kernel to the linker and CBD area in a suitable coding DNA using well known recombinant DNA technology for the production of the desired recombinant protein. In short, polynucleotide, encoding the areas of mergers, amplified and clone; nucleotides can be synthesized. Flushed polynucleotide inserted into the expression vector, the vector transform the cell the owner and Express the protein. Preferably, the linker and CBD is attached to the C-end endoglucanases kernel.

The expression vector is a cloning plasmid or vector that can Express the DNA encoding endoglucanase proteins merger, after the introduction of the vector into a suitable cell host. When using fungi as hosts gene of interest is preferably introduced into the fungal cell host comprising cloning or expressing the vectors integrated into the fungal chromosome or allows integration of the gene of interest into the chromosome of the host. Other sequences that are part of a cloning or expressing the vectors can also be integrated together with the indicated DNA integration process. In addition, the fungi cells expressing vector or part thereof may be directed in a predetermined locus. Alternatively, the desired gene fusion can be represented as Autonomous plasmids can replicate.

DNA encoding endoglucanase fusion proteins, preferably placed under the control of (i.e., the functionality is however associated with certain regulatory sequences, such as the promoter sequence in the vector. During the transformation of these regulatory sequences integrate into the host genome along with the gene of interest. Alternatively, the regulatory sequences can be embedded in the site of integration.

Controlling the expression sequence comprising expressing vector vary depending on, whether designed vector for expression of a particular gene in a prokaryotic or eukaryotic host (for example, vector-vector may contain a gene for selection in bacterial host). Sequence controlling the expression may contain regulatory elements for transcription, such as promoters, enhancers, and the sequence termination of transcription and/or regulatory elements of the broadcast, such as sites of translation initiation and termination.

Polynucleotide molecule, such as DNA, is able to Express a polypeptide if it contains a sequence controlling the expression includes information on the regulation of transcription and functionally linked to a nucleotide sequence that encodes a polypeptide.

The functional relationship is a relationship in which the sequence is connected with the regulatory sequence (or after what euteleostomi) thus, that expressing the sequence is in this area where exposed to the influence or control of the regulatory sequence. Two DNA sequences (such as a promoter sequence of the plot associated with the 5'-end sequence that encodes a protein) are considered to be related functional way, if the promoter affects the transcription.

Vectors in accordance with the present invention can contain other functionally linked regulatory elements such as enhancer sequences.

In a preferred embodiment of the invention construct genetically stable transformants so that DNA encoding fusion proteins inserted into the chromosome of the host by transformation with a vector, which may contain sequences that allow the integration of the specified vector in the chromosome.

Cells, chromosomes are stably integrated DNA encoding endoglucanase fusion proteins, can be selected, for example, through the inclusion of the marker (marker), homologous or heterologous, which provides the selection of host cells containing the expression vector in the structure of chromosomes. The marker may provide resistance to biocides, for example, resistance to antibiotics or heavy m is the tall, such as copper. In addition, the markers can complementarity auxotrophic mutation in the chromosome of the host, etc. of Breeding token can represent, for example, breeding gene directly linked to a DNA sequence, which is the subject of the expression of the gene, or it can be entered into the same cell by co-transformation. Can be used by other system selection.

After constructing the expression vector containing DNA encoding the protein of the merger, it is introduced into a suitable cell host any suitable way, including transformation, known from the prior art. After transformation of recipient cells are grown in an appropriate selective medium, which selects the transformed cells for their growth.

Suitable host systems for the expression and production of the protein are, for example, the production system on the basis of the fungi Trichoderma (EP 244 234) or Aspergillus such as A. oryzae or A. niger (WO 97/08325, WO 95/33386, U.S. patent 5843745, U.S. patent 5770418), or system of products on the basis of the fungi Fusarium, such as F. oxysporum (Malardier et al., 1989). Suitable systems are products based on bacteria include system based on Bacillus, for example, B. subtilis, B. licheniformis, B. amyloliquefaciens, or E. coli or Streptomyces.). Suitable production system based on yeast include systems based on Saccharomyces, Shizosaccharomyces is whether Pichia pastoris. Can be used and the production system on the basis of other microorganisms or mammalian cells or plants.

Expression of the cloned gene sequence leads to the production of the desired protein or protein fragment. This expression can occur constantly in transformed cells or in a controlled way.

To obtain the enzyme preparations according to the claimed invention, the hosts having the desired properties (owners, are able to Express economically significant quantities endoglucanase proteins merger), cultivated in suitable conditions, and the desired enzymes preferably are secreted from the host body into the culture medium. Then proteins, if necessary, allocate the specified culture medium by known methods. Preferably, the owners for such production are filamentous fungi such as Trichoderma or Aspergillus, in particular, T. reesei.

Used herein, the term "enzyme preparation" refers to any enzyme product that contains at least one of the stated endoglucanase proteins merge. Thus, the enzyme preparation can be an emaciated culture medium or filtrate. Emaciated cultural environment means an environment in which it was cultivated by the owner, and which has produced the household is different enzymes. Preferably, the cells of the host separated from the specified culture medium after producing enzymes. If necessary, the enzyme preparations can be dried or enzyme activity may be concentrated and/or stabilized to save. If required, the desired enzyme may be further purified in accordance with known suitable methods such as extraction, precipitation, chromatography, affinity chromatography, electrophoresis, and other similar methods.

However, an advantage of the claimed invention lies in the fact that cultural environment, whether or not containing host cells, can be used as an enzyme preparation as such without further purification, since endonuclease fusion proteins can secretariats into the culture medium and to be active in this culture medium. Enzyme preparations are extremely economical from the point of view of their production and consumption, and the allocation of specific enzyme from the culture medium is not required.

In addition to endoglucanases the fusion protein, enzyme preparations can contain one or more other enzymes, which may be, for example, other cellulase, amylase, lipase, protease, hemicellulase, xylanase, pectinase and/or is xidase such as laccase and peroxidase. Alternatively, before, during or after processing endoglucanase protein fusion can be processed by another enzyme. Processing enzyme may include, for example, processing one or more amylase (for example, for rasshifrovka denim), or one or more hemicellulase, or one or more peroxidase, or one or more lactase. It depends on what enzymes are enzyme preparation or used for processing.

In addition to the fusion protein, the enzyme preparation may contain various additives such as stabilizers, buffers, preservatives, surfactants and/or components of the culture medium. Preferred additives are additives that are commonly used in enzyme preparations for certain purposes.

Enzyme preparations can be obtained in liquid or solid form, for example, in the form of a dry powder or granules, in particular, in the form of stable granules and stable liquid. It should be borne in mind that the enzyme preparations can be further enriched, or partially or completely deprived of certain specific enzyme activity in order to meet the requirements of the specific use in various cases, for example, in the textile industry. The mixture of enzymes with different activity, critirea master, may have an advantage in certain industrial applications, for example, in biostoning and biofinishing.

Endoglucanase proteins mergers and preparations containing such proteins, are used, for example, for the production of textiles, food and feed products, vegetable oils, cellulose pulp and paper industry. These proteins can be used to handle any cellulose-containing material such as textile material, plants used in animal feed, plant material for extraction of oil, cellulose-containing mass, obtained from wood by mechanical or chemical treatment, or secondary fiber. They can also be used in detergents, which normally contain additional components, for example, surface-active substances, surfactants, bleaches and/or fillers. In the context of the present invention, the term "cellulosic material" refers to any material containing cellulose or its derivatives as a main component. Cellulose-containing material is introduced into contact with an effective amount of fusion protein in suitable conditions, for example, under appropriate pH and temperature, and provide a response within the time required for it.

Fused enzymes especially Olesky for the treatment of textile materials, such as fabrics and clothing (clothing items). Textile materials can be produced from natural cellulose fibers or of man-made cellulose fibers or mixtures thereof, or of a mixture of synthetic fibers and cellulosic fibers. Preferably, the cellulosic material is a cotton, especially denim. The term "denim" in the context of the present invention relates to fabrics of denim, especially clothes made of denim, including jeans. Preferably, denim is a material dyed Indigo. Denim can also be processed derivatives of Indigo or Indigo together with other dyes, for example, denim dyed with Indigo together with sulfa possible.

Endoglucanase fusion proteins are particularly useful for biostoning and biofinishing.

Washing with pumice stones consists of three phases: rasshifrovka, abrasive processing and final processing. The first stage typically includes a wet processing of jeans and accompanied by the removal of starch and other dressing material for agents commonly used for treatment of the fundamentals of the yarn to prevent damage during weaving. Alpha-amylase is used to remove the dressing material for agents on the basis of starch to improve and versatility of the process of wet processing. After rasshifrovka jeans usually is otpolaskivayut in water or subjected to direct abrasive processing.

The second stage, blasting, is carried out using enzymes or pumice stones or both of these means. In all cases, removal of dye necessary mechanical effects and the treatment is usually carried out in washing machines, and similar machines with reels. The term "aged" ("cooked") refers to the appearance of denim fabric that has been processed cellulase enzymes or pumice stones or both of these means. Synonyms for this term are the terms "worn look" and "type of washing with stones". Due to uneven removal of the dye, there is a contrast between the colour and the areas from which the dye has been removed.

For abrasive machining followed by a third stage, post-processing, which involves the stages of washing and opolaskivanija, in the process which can be used in detergents, optical brighteners, whitening agents or emollients. After processing by the enzyme reaction is stopped to prevent damage to the treated material, for example, the inactivation temperature and/or pH, during which the final laundering and/or removal of detergents This means that the mechanical strength is not already exposed to enzymes.

Used herein, the term "biostoning" TKA and or service means the use of enzymes instead, of, or in addition, to the use of pumice stones for processing of fabric or clothing, especially denim.

As noted above, treatment with cellulases can completely replace washing with pumice stones (for example, 1 kg of commercial enzyme replaces the use of 100 kg of stones). However, treatment with cellulases can be combined with treatment with pumice stones, when you need to get very worn product. The effect of peach skin, when an external appearance resembling a thin speaker fuzz peach, also achieved a combined washing with a neutral cellulase and pumice stones. Described fusion proteins are particularly useful for the effective delivery shabby appearance and minimize back staining during biostoning.

Biostoning usually carried out at a pH of from 3.0 to 8.0, preferably at pH from 5.0 to 7.0. The temperature may vary from about 30°C to 80°C and, preferably, from 50°C to 60°C. the ratio of the volume of liquid to the weight of the fabric can vary from about 3:1 to 20:1, preferably from 5:1 to 10:1. Processing time varies from 15 min to 90 min, preferably from 30 minutes to 60 minutes Should be borne in mind that the dose of enzyme will depend largely on the type of fabric, washing machines, washing conditions (pH, temperature, the ratio of liquid volume to weight of fabric, load denim, the speed of the process is (a) and the type of enzyme preparation, etc. If you want, you can also use pumice stones in combination with endoglucanase fusion proteins. The dose of enzymes can be significantly reduced. The person skilled in the art understand how to choose the right dose of enzymes and washing conditions.

Declared endoglucanase fusion proteins provide unexpected advantages, because the enzymes have high activity with minimal reverse staining of tissues and a good contrast between the painted and unpainted areas. In addition, fusion proteins are easily obtained and they can be used at relatively high variation of temperature and pH.

Endoglucanase fusion proteins are useful for biofinishing fabrics and clothing. Biofinishing refers to the use of enzymes in the process of the controlled hydrolysis of cellulose fibers for surface modification of fabrics or yarns thus, in order to prevent permanent rolling, to improve the softness and smoothness of the fabric, when it is tried to the touch, clean the surface structure with the removal of the spool, resulting in a clearer color, improves the drape of the fabric, moisture absorption and the absorption of the dye.

Enzyme, defiling can be carried out at any stage wet processing of fabric, preferably, after optional rasshifrovka and/or bleaching under conditions which, similar to conditions of biostoning.

Used endoglucanase fusion proteins, including beta-glucanase, hemicellulase, or xylanase can be useful for improving the quality of animal feed, particularly for the processing of plant material by enzymes.

Starch, proteins and lipids can easily be broken down in the digestive system of monogastric animals such as poultry and pigs, while a large part of non-starch polysaccharides (NSP), including associated mixed beta-glucan, for example from barley and oats, remain unsplit, because these animals lack the enzymes with the desired activity. Moreover, the digestibility of other components, particularly animal fats, is reduced in the presence of NSP.

Beta-glucanase commercially used for solving problems related to their associated mixed beta-glucanes from barley and oats. It is known that beta-glucanase reduce the viscosity of the intestinal contents, caused by soluble beta-glucans, and release nutrients encapsulated cell walls rich in beta-glucans. The use of beta-glucanase improves the health of animals, as determined by weight gain and the ratio of processing food. In addition, the house is her birds decreases the stickiness of the litter.

Granulation using steam is the main technological procedure of cooking food in the world. The advantages of its products from the food mass is more easy handling, reduction of toxic components and organisms in the aforementioned improvements in the quality of the product. Thermostability and high activity of the fusion proteins described above make them suitable for use in food production.

The invention is illustrated by the following non-limiting examples. It should be understood that embodiments of the invention described in the description, as examples, are presented solely for illustrative purposes and the scope of the invention includes various changes and modifications.

Example 1. The production of cellulase Thermoascus aurantiacus ALKO4242 EG 28 in Trichoderma reesei

For isolation and enzymatic treatment of DNA (plasmids, DNA fragments, transformation of E. coli, etc. using standard biological techniques. Used the basic methods described in the manuals of molecular biology, for example, the manual Sambrook et al. (1989) and Sambrook and Russel (2001).

Cel5A gene Thermoascus aurantiacus (SEQ ID NO:1)encoding the cellulase EG28 (SEQ ID NO:2), amplified by PCR directly from genomic DNA Thermoascus aurantiacus ALKO4242 allocated by the RV method and Broda (1985. Direct (SEQ ID NO:9) and reverse (SEQ ID NO:10) primers designed based on the published sequence endoglucanase Thermoascus aurantiacus (AF487830). Amplificatory product size 1.3 KB containing the complete gene (from start codon to the stop codon), clone in the form of a SacII- > PST fragment in the vector pBluescript II KS+. Is sequenced two independent clone and select one clone, which indicate pALK.1926. The E. coli strain containing pALK1926 deposited in the German collection of microorganisms and cell cultures GmbH under the number DSM 17326.

Plasmid expression (pALK1930, 1) design for the production of recombinant cellulase Thermoascus aurantiacus EG28/Cel5A (SEQ ID NO:2). Cel5A gene, including its own signal sequence, just merge with the cbhl promoter Trichoderma reesei using PCR. The cbhl promoter, terminator cbhl and amdS marker gene included in the plasmid, as described Paloheimo et al. (2003). A linear expression cassette (Figure 1) was isolated from the backbone vector by cleavage with restriction enzymes, introduced by transformation into Trichoderma reesei A96, and select transformants using ndimethylacetamide as the sole source of nitrogen. The host strain devoid of the four major endogenous cellulases: CBHI/Cel7A, CBHII/Cel6A EGI/Cel7B and EGII/Cel5A. Transformants are grown in accordance with the methods Penttilä et al. (1987) with modifications described Karhunen et al. (1993) purified Transformants on selective tablets to the stage of conidia and str is ulali on PD.

Production of cellulase by transformants analyzed in the culture supernatant under cultivation in shake flasks (50 ml). Transformants grown for 7 days on a complex environment with cellulose (Joutsjoki et al. 1993), buffered with 5%KH2PO4at pH 5.5. Enzymatic activity of the recombinant protein was measured in the culture supernatant by the release of reducing Sugars from carboxymethyl cellulose (2% CMC) at 50°C in 50 mm acetate buffer (pH of 4.8), as described by Bailey and Nevalainen, 1981; Haakana et al. 2004. Activity against beta-glucan barley (1%) is also determined by measuring the release of reducing sugars 50°C in 50 mm acetate buffer (pH of 4.8), as described Stálbrand et al. 1993. Production of recombinant protein is also determined in the culture supernatant by SDS-polyacrylamide gel electrophoresis. The genotypes selected transformants analyze southern-blotting using the expression cassette as a sample.

The optimum pH heterologic produced cellulase EG28/Cel5A define universal Mcllvaine buffer at pH range from 4.0 to 8.0 using carboxymethylcellulose as a substrate. As shown in Figa, the optimum pH for activity of cellulase EG28/Cel5A is 6.0. The optimum temperature of the enzyme activity of cellulase EG28/Cel5A is 75°C (Pigv).

Display the config of transformant RF6188 cultivated in the bioreactor with obtaining material for testing possible applications (see Example 4).

Example 2. The preparation of recombinant fusion protein Thermoascus aurantiacus ALKO4242 EG28+CtCBD

To obtain recombinant protein fusion Thermoascus aurantiacus ALK04242 EG 28+CtCBD (SEQ ID NO:4) pulp-binding domain (CBD) of cellulase CBHI/Cel7A Chaetomium thermophilum ALK4265 connect with cellulase EG28/Cel5A. The construct containing the catalytic domain EG28 (amino acids 1-334 full-length polypeptide), attached to the linker and CBD Chaetomium thermophilum CBHI/Cel7A CtCBD (SEQ ID NO:15).

Using standard molecular biological methods described in Example 1. First embed a unique restriction site SnaBI about C-terminal segment EG28/Cel5A by PCR. This provides a direct merge any DNA with blind ends after amino acids Y334 polypeptide EG28/Cel5A. The linker and CBD-domain CBHI/Cel7A Chaetomium thermophilum encoding gene (Cel7A, SEQ ID NO:17) amplified by PCR using the forward (SEQ ID NO:11) and reverse (SEQ ID NO:12) primers and genomic DNA Chaetomium thermophilum ALK4265 as a matrix. Amplificatory 1.6 KB product are ligated with the gene Cel5A (after Y334) obtaining The Cel5A_Ct Cel7CBD (SEQ ID NO:3). The obtained plasmid denote pALK1946. The strain of E.coli containing pALK1946 deposited in the German collection of microorganisms and cell cultures GmbH under the number DSM 18159.

Plasmid expression (pALK1948, Figure 1) to obtain cellulase EG28+CtCBD design, as described in Example 1. Linear tapes the size of 8.9 KB (Figure 1) was isolated from the backbone vector by cleavage with restriction enzymes Notl, enter the transformation in Trichoderma reesei A33 (strain devoid of genes encoding four main endogenous cellulase: CBHI/Cel7A, CBHII/Cel6A EGI/Cel7B and EGII/Cel5A) and select the transformants as described in Example 1. The optimum pH and temperature for protein merger define as well as for the wild-type protein EG28.

The selected transformant RF6377 cultivated in the bioreactor with obtaining material for testing possible applications (see Examples 5-10).

Example 3. The preparation of recombinant fusion protein Acremonium thermophilum ALKO4245 EG40+CtCBD

To obtain recombinant protein fusion Acremonium thermophilum ALKO4245 EG40+CtCBD (SEQ ID NO:6) pulp-binding domain (CBD) of cellulase EG40 replace those Chaetomium thermophilum ALKO4265 CBHI/Cel7A. The construct containing the catalytic domain EG40 (amino acids 1-234 full-length polypeptide), attached to the linker and CBD Chaetomium thermophilum (CtCBD, SEQ ID NO:15).

Using standard molecular biological methods described in Example 1. Gene Cel45A Acremonium thermophilum (SEQ ID NO:8) amplified by PCR from genomic DNA of Acremonium thermophilum ALKO4245 using primers (SEQ ID NO:13) and (SEQ ID NO:14). The PCR fragment of 1.1 KB clone in the vector pBluescript II KS+ as SacII fragment- > PST. Then embed a unique restriction site NruI about-To-end area EG40 by PCR. This provides a direct merge any DNA with blind ends after amino acids S234 polypeptide is EG40. The linker and CBD-domain CBHI/Cel7A Chaetomium thermophilum encoding gene (Cel7A) amplified by PCR as described in Example 2. Restriction fragment are ligated with the gene Cel45A (after S234) obtaining At Cel45A_Ct Cel7CBD (SEQ ID NO:5). Next, construct the expression plasmids for the production of cellulase EG40+CtCBD and produce recombinant protein (SEQ ID NO:6) in Trichoderma, as described in Example 1.

Example 4. The activity of cellulase EG28 in final processing of denim at different temperatures

The cellulase Thermoascus aurantiacus EG28 obtained in Trichoderma (strain RF6188), as described in Example 1 are tested for the ability to create a worn look denim (biostoning), which is provided by traditional washing with pumice stones, at different temperatures. As a control comparison using effective enzyme for final processing of denim, representing commercial drug EGII enriched acidic cellulases obtained in Trichoderma (U.S. patent 5874293).

As the test material used jeans, made of dyed Indigo denim, after rasshifrovka alpha-amylase ECOSTONE®A200. Treatment with cellulase is carried out in the washing machine Electrolux Wascator FOM 71 CLS under the conditions described in Table 1.

Endoglucanase activity (ECU) preparation of enzymes is determined by the release of reducing sugars from hydrocellulose, the AK previously described by Bailey and Nevalainen, 1981. The drug EGII enriched acidic cellulases, and EG28 dispense as 220 ECU/g and about 380 ECU/g tissue, respectively. Enzymes are tested at their optimum pH, namely the drug EGII at pH 5 and the drug EG28 at pH 6. After drying inactivate enzymes (10 min at 40°C) raising the pH to 11 with sodium hydroxide. Then jeans three times otpolaskivayut water and dried in the dryer.

The effect of biostoning/level of wear is determined by measuring color values reflected in the spectrophotometer Minolta CM 2500 when using the color scale coordinates L*a*b* (illumination D65/2°). The intensity of the color of the front and back sides of the denim pockets and measured after rasshifrovka (i.e. before treatment with cellulases) and after treatment with cellulases. Each dimension of the color of the front and back sides of the denim pockets and represent the average of about 40, 20 or 12 measurements, respectively. For each test using two pairs of jeans and the final result is presented as an average value. The results of the experiments are shown in Table 2 and figure 3

Table 1.
The test conditions of cellulases during processing denim
The parameters of the testing process
Download denim1.3 kg
Water19 l
The pH control (pH 5-5,3)
The pH control (pH of 6.1 and 6.3)
35 g of Na2HPO42H2O+22 g of citric acid
35, 5 g of Na2HPO42H2O+15 g of citric acid
Time45 min
Temperature40, 50, 60 or 70°C
Dose of cellulase220 or 380 ECU/g tissue

The results are shown in Table 2 and figure 3 suggests that the best effect of wear when using the drug EG28 (strain RF6188), is provided at a temperature of from 50 to 70°C. When using a dose of 380 ECU/g tissue preparation EG28 at 70°C observed a similar level of attrition (loss of color L*) when compared with using a dose of 220 ECU/g tissue preparation EGII at 60°C. However, the effect of reverse painting (redistribution of Indigo dye) on the reverse side of the denim pockets and below when using the drug EG28 (higher the loss of color, less blue) when compared with the use of the drug EGII. Also the contrast color on the face side of denim is more pronounced.

Example 5. And the activity of cellulase fusion protein EG28+CtCBD compared to cellulase EG28 in final processing denim

Recombinant protein fusion Thermoascus aurantiacus EG28+CtCBD obtained in Trichoderma (strain RF6377), as described in Example 2, compared with the drug EG28 from strain RF6188 in the process of biostoning denim at pH 6 and a temperature of 60°C. Wash and testing system for biostoning the same as in Example 4, except that the weight of the denim reaches 1430 g, together with various additional pieces of denim, which are not included in the measurement. The effect of treatment with cellulases evaluated as in Example 4.

The results are shown in Table 3, indicate that the effect of biostoning drug EG28+CtCBD is very good at low dose. When comparing products strain RF6377 and strain RF6188 observed a similar effect of wear (loss of color L*), but this requires 6.5 times lower dose product RF6377 compared with the product RF6188. Accession pulp-binding domain (CBD) to the cellulase EG28 does not increase the undesirable effect of reverse painting, but significantly increases the activity directed on abrasion (digestion) of tissue.

Table 3.
Measurement of color fabric Denny processed EG28 and EG28+CtCBD by cellulases, at pH 6 and a temperature of 60°C
* indicates a loss of color, -b* blue, +b* yellowness.
EnzymeECU/g serviceBefore treatment with cellulaseAfter treatment with cellulaseDelta L*Delta b*
L*b*L*b*
Facial party
EG2832523,40-16,0231,01-17,12a 7.62-1,10
EG28+CICBD5023,81-16,2131,60-16,927,80-0,71
The reverse side
EG2832549,46-6,8350,03-9,620,57-2,79
EG28+CICBD5049,36-7,0350,62-9,611,27-2,58
Pockets
EG2832576,11-8,3668,53-12,46-7,58-4,10
EG28+CICBD5076,028,4568,81 -12,39-7,21-3,94

Example 6. The activity of the cellulase fusion protein EG28+CtCBD compared with EGII cellulase in final processing denim

Recombinant protein fusion Thermoascus aurantiacus EG28+CtCBD obtained in Trichoderma (strain RF6377), as described in Example 2, compared with the commercial drug EGII enriched acidic cellulases (U.S. patent 5874293), when biostoning denim.

The judging system for biostoning the same as in Example 4, except that use pieces of different types of denim Ukos Sport (Belgium) and Vicunha (Brazil) with a total weight of 1, 2 kg. of processing Conditions, EG28+CtCBD and EGII are shown in Table 4. The effect of treatment with cellulases evaluated as in Example 4.

The results of experiments on the processing of four different types of denim are shown in Tables 4 and 5. When using doses of 500 ECU/g tissue preparation EG28+CtCBD observed a similar level of wear when compared with using a dose of 1000 ECU/g tissue preparation EGII. When using the drug EG28+CtCBD requires only about half the ECU-dose EGII enriched acidic cellulases. In addition, for the process of biostoning cultural environment in which it was grown recombinant host, producing EG28+CtCBD, volume twice as effective environment in which it was grown recombinant the second host, producing EGII. Accordingly, an inverse coloration to a lesser extent, was observed on the reverse side of the denim when using the drug EG28+CtCBD. The activity of the drug EG28+CtCBD at 50°C was best at pH 6 than at pH 5.

Example 7. The effect of pH on cellulase activity of the drug EG28+CtCBD at 50°C and 60°C

Recombinant protein fusion Thermoascus aurantiacus EG28+CtCBD obtained in Trichoderma (strain RF6377), as described in Example 2 are tested for activity at biostoning denim at 50°C and 60°C.

Dyed Indigo denim (different from the one used in the preceding examples) after rasshifrovka and cut into pieces to use as test material. Treatment with cellulase is carried out in the device LP-2 Launder Ometer, as described below. About 7.2 g of tissue denim (flap about the size of 12 cm × 12 cm), 200 ml of citrate-phosphate buffer Mcllvaine and 90 steel balls (diameter 0.6 cm) load 1, 2 l containers. The device Launder Ometer runs for 120 min at different pH values from 4 to 9 and a temperature of 50°C and 60°C. After removing the flaps from their containers otpolaskivayut water and soaked in water containing NaOH (pH 11) for 10 min under stirring. Then rags soaked in warm water containing liquid detergent (OMO Color) and stirred for 10 min and then OS is Orono otpolaskivayut warm water several times. The shreds are dried at room temperature. The effect of treatment with cellulase evaluated as in Example 4. Each dimension on the front side of the flap denim is defined as the average of 20 measurements.

The results of the experiments are shown in Tables 6 and 7 and figure 4 and 5 show that the optimum pH for cellulase EG28+CtCBD at 50°C range from 5.5 to 6.5 and at 60°C from 5 to 7.

Table 6.
Measurement of fabric colors on the face side of denim treated with cellulase EG28+CtCBD, at different pH in the device Launder at 50°C
L* indicates a loss of color, -b* blue.
EnzymeECU/g serviceConditionsBefore treatment with cellulaseAfter treatment with cellulaseThe increase of L*
L*b*L*b*
The buffer control0pH 6 17,28-13,0718,77-14,131,49
The buffer control0pH 817,01-13,418,18-14,451,17
EG28+CICBD1000pH 416,79-13,5920,86-16,404,07
EG28+CICBD1000pH 517,34-13,1821,59-16,444,25
EG28+CICBD1000pH 5.517,30-13,25representing 22.06-16,394,76
EG28+CICBD1000pH 6the 17.3 -13,4222,29-16,474,94
EG28+CICBD1000pH 6.517,31-13,4122,48-16,655,17
EG28+CICBD1000pH 717,34-13,2521,55-16,234,21
EG28+CICBD1000pH 816,01-13,6319,62-15,722,71

Table 7.
Measurement of fabric colors on the face side of denim treated with cellulase EG28+CtCBD, at different pH in the device Launder at 60°C
L* indicates a loss of color, -b* blue.
Ermentau medication ECU/g serviceConditionsBefore treatment with cellulaseAfter treatment with cellulaseThe increase of L*
L*b*L*b*
EG28+CICBD1000pH 417,19-13,5820,48-15,773,29
EG28+CICBD1000pH 517,10-13,2422,74-16,595,64
EG28+CICBD1000pH 5.517,10-13,54cushion 22.66-16,615,56
EG28+CICBD1000pH 617,10 -13,65to 22.83-16,745,73
EG28+CICBD1000pH 6.517,19-13,4722,65-11,725,46
EG28+CICBD1000pH 716,99-13,7222,10-16,665,11
EG28+CICBD1000pH 817,13-13,6819,35-15,152,22
The buffer control0pH 617,36-13,618,95-14,281,59

Example 8. The cellulase activity of the drug EG28+CtCBD in final processing of denim different temperatures

Recombinant protein clean what I Thermoascus aurantiacus EG28+CtCBD, received in Trichoderma (strain RF6377), as described in Example 1 are tested for the ability to create shabby fabric, similar to that provided in the wash denim with pumice stones, at different temperatures. As a control comparison using efficient enzymes for final processing of denim, namely commercial drug EGII enriched acidic cellulases (U.S. patent 5874293), and the drug on the basis of neutral cellulases ECOSTONE®C1. The judging system for biostoning the same as in Example 4, except that use pieces of different types of denim and the washing time is 55 minutes of Enzyme activity (endoglucanase unit, ECU) drugs EG28+CtCBD and EGII determined as described in Example 4. Activity (unit neutral cellulase, NCU) of the drug on the basis of neutral cellulases ECOSTONE®C1 is determined by the release of reducing sugars from carboxymethyl cellulose, as described by Bailey and Nevalainen, 1981; Haakana et al. 2004. Dose ECOSTONE®C1, EG28+CtCBD and EGII is 250 NCU/g tissue, 500 ECU/g of tissue or 1000 ECU/g tissue, respectively. The effect of treatment with cellulase evaluated as in Example 4.

Activity EG28+CtCBD at a temperature of from 40 to 70°C and pH 6 are shown in Table 8 and figure 6. The preferred temperature range for the enzyme ranges from 50 to 70°C. lower temperatures such as 40 is C, can be used to obtain the effect of rubbing with greater color intensity, if it is desirable that the fabric looked darker.

The research results are consistent with those obtained in Example 6, where the activity of EG28+CtCBD when biostoning approximately two times higher than the activity of the commercial drug EGII enriched acidic cellulases. Acid cellulase usually contribute reverse staining. Neutral cellulase, such ECOSTONE®C1, typically called a weak inverse staining, providing a good contrast. The results obtained in this example show that a similar effect is detected when using cellulase EG28+CtCBD and neutral cellulases (table 8).

As a control comparison using commercial drug EGII enriched acidic cellulases, and preparation on the basis of neutral cellulases ECOSTONE®C1. L* indicates a loss of color, -b* blue, +b* yellowness.

Example 9. The activity of cellulase preparations EG28 and EG28+CtCBD when biofinishing (removing the spool)

Testing the activity of cellulase EG28 from strain RF6188 and drug EG28+CtCBD from strain RF6377 when removing the spool from cotton Jersey. Treatment with cellulases carried out in the washing machine Electrolux Wascator FOM 71 CLS under the conditions described in Table 9.

Flaps two lower the th quality sweaters with collared "Polo", on the surface of which there are numerous pellets, made of 100%cotton fabric type Jersey or 95%cotton fabric type reps and 5%lycra used as the test material and control. The samples are first pre-washed for 10 min at 60°C and 1 ml/l of a surfactant/wetting agent (Sandoclean PCJ, Sandos, and Imacol CN, Clariant) and three times otpolaskivayut water. After that cotton knitted fabric is treated with cellulase at 60°C for 60 min in the presence of the same textile additives, which are used during the preliminary washing. The inactivate enzymes as described in Example 4, except that the temperature during alkaline opolaskivanija is 60°C. the Flaps Jersey three times otpolaskivayut and dried in the dryer.

Table 9.
The test conditions of cellulases in final processing (biofinishing) denim
The parameters of the testing process
Download fabric1.0 kg
Water15 l
Sandoclean PCJ and Imacol CN1 ml/l
Control pH/buffer (pH 5-5,3)approximately 3 ml of acetic acid (80%)
Time60 min
Temperature60°C
Dose of cellulase1200 ECU/g tissue

The effect of treatment with cellulases assessed visually by the naked eye and with a magnifying glass. As a control, use pre-washed tissue samples without enzyme.

The results of the experiments show that the drug EG28+CtCBD increases the resistance of the tissue to the slide. The number of the spool on the surface of the knitted fabric is significantly reduced after treatment EG28+CtCBD compared with control (washing without enzyme). The drug EG28 also has a similar effect.

Example 10. Test the stability of the granulated food product

Two cellulase preparation EG28 from strain RF6188 and drug EG28+CtCBD from strain RF6377 individually tested in an experiment reminiscent of the industrial method of production of the food product. Before testing drugs EG28 and EG28+CtCBD, spray dried, ground with wheat flour to improve the homogeneity. Enzymes EG28 and EG28+CtCBD, pre-mixed with the flour, add in doses of 200 g/ton 500 g/ton of food, appropriate to estwenno. Overdose enzymes are used to support the analysis of samples of granular at high temperatures, when their activity can be significantly reduced.

To obtain food mixture in the process of pilot-scale production of vegetable food product with a nominal capacity of granulation 5 t/h use the mill and mixer. The mill is a unit Champion, equipped with a grid of holes with a diameter of 3.0 mm Primed raw material mixed in 2,000 l horizontal mixer at 27 rpm prior to placement in the device food the mini. The device contains a horizontal mixer (volume 700 l, speed 48 rpm, the mixing capacity 300 kg)dosing screw Skjold TR and cascade mixer Kahl (length 130 cm, diameter 30 cm, speed of 155 rpm), equipped with a 37 moving pallets. The time interval to 300 kg/h is about 30 C. On the side of the cascade mixer mounted manifold with a starting device for water and three steam Ventes for the supply of steam to the food. Steam is generated in the boiler under high pressure with a maximum capacity of 400 kg of steam/hour. Testing is carried out at a pressure of 2 atmospheres and the steam is supplied from ventile by reducing the pressure under the control of the introduction of a pair of cascaded mixer Three ventila on the collector is used to produce the desired temperature of the food is the same time. The temperature of the food product is measured with a digital thermometer located at the outlet of the cascade mixer, just before the product gets to the net for granulation. Use press for granulation Simon Heesen type monologo with mesh diameter of 3 mm and a motor of 7.5 kW. Internal mesh diameter of 173 mm, the height of the press roller 50 mm, the diameter of the press roller 140 mm, rotation rate of 500 rpm and the rated capacity 300 kg/h Samples taken after they were granulated in the press and cooled in cloisonne cooling container with a perforated bottom at a speed of ventilation 1500 m3air/h

Get a batch of wheat mass weighing 300 kg of Premix obtained from 10 kg of the specified product and 200 g (or 500 g) of the tested enzyme in 70 l in the mixer. The rotation speed of the mixer 45 rpm and mixing time of 10 minutes Specified premix is added to 290 kg of food product and placed into a horizontal mixer device food of mini and stirred for 10 minutes After sampling the formed mass of food product granularit passing through the press (the diameter of the holes of the mesh 3 mm). The product is heated to the desired temperature (from 65 to 90°C) by the introduction of steam in a cascade mixer. For each temperature of the first sample taken after 10 min after reaching the desired temperature granulation (which is th measured in the food product just before granulation). Samples taken during 1,0 min, which corresponds with 5.0 kg of granulated food product. Sub-sample weighing about 500 g is placed in a cooling container for 10-15 after the pellets come out of the press. All samples aeronaut and cooled at a similar temperature for 15 minutes the Samples uniformly divided into special dividing device and fill their plastic bags.

Samples analyzed as follows: 2.5 g well primed sample and 20 ml of acetate buffer (0.05 M, pH 5.0) was stirred at room temperature for 30 min, centrifuged (10 min, 4000 rpm) and diluted. 1.0 ml of purified sample in three repetitions balance for 5 min in a water bath at 40°C. the Reaction is initiated by adding tablets beta glucosami (Megazyme, Ireland) without stirring. After 30 min the reaction is stopped by the addition of 5.0 ml of Trizma Base, 1% (weight per volume) (Sigma-Aldrich) with intensive stirring vortex. The test tube and leave at room temperature for approximately 5 min; the liquid is stirred again and filtered through filter paper Whatman I. Absorption of the filtrate was measured at 590 nm. The results are analyzed using the standard curve.

Enzyme activity determined to granulation and after granulation. The data presented in Table 10. The influence of the temperature of the granulation on the activity of the beta-glucanase is also shown in Fig.7 and 8. Cellulase EG28 and EG28+CtCBD stable at temperatures up to 80°C. Cellulase EG28+CtCBD more stable than EG28 at a temperature of granulation 90°C.

Table 10.
The activity of enzymes to the food product from the horizontal mixer) and after granulation at a temperature of 65°C to 90°C
The temperature of the granulationThe activity of beta-glucanase (FBU/kg food)
(EG28)(EG28+CtCBD)
A food product of the horizontal mixer239000±9000 (100%)1148000±48000 (100%)
Granulation (65°C)227000±16000 (95%)1007000±67000 (88%)
Granulation (70°C)217000±5000 (91%)1012000±54000 (88%)
Granulation (75°C)227000±14000 (95%)993000±49000 (86%)
Granulation (80°C)215000±6000 (90%)1030000±87000 (90%)
Granulation (85°C)188000±7000 (79%)904000±56000 (79%)
Granulation (90°C)149000±8200 (62%)861000±32000 (75%)

A list of the deposited organisms

StrainContained a plasmidBody escrowDate DepositRoom Deposit
Acremonium thermophilum ALKO4245-CBS(1)September 20, 2004CBS 116240
Thermoascus aurantiacus ALKO4242-CBS(1)September 20, 2004CBS 116239
Chaetomium thermophilum ALKO4265-CBS(2)November 8, 1995CBS 730.95
Escherichia colipALK1946DSMZ(3)April 7, 2006DSM 18159
Escherichia colipALK1926DSMZMay 13, 2005DSM 17326
(1)Central collection of cell cultures in Uppsalalaan 8, 3584 CT, Utrecht, NL
(2)Central collection of cell cultures in Oosterstrat 1, 3742 SK BAARNt, NL
(3)German collection of microorganisms and cell cultures GmbH (DSMZ), Mascheroder Weg, 1b, D-38124 Braunschweig, DE

1. Cellulose protein containing the first amino acid sequence endoglucanases engine having at least 95%identity with SEQ ID NO:2 and cellulase activity, or a fragment of a specified protein having cellulase activity and a second amino acid sequence containing the linker and cellulose-binding domain (CBD)that has at least 95%identity with SEQ ID NO:15 and cellulose-binding activity, or a fragment of a specified protein with cellulose-binding activity.

2. Fused protein according to claim 1, where the endoglucanases belongs to the family of 5 glycosylglycerols.

3. Protein according to any one of claims 1 or 2, where endoglucanases the core comes from Thermoascus aurantiacus, particularly So aurantacus CBS 116239.

4. Fused protein according to claim 1, where endoglucanase core has at least 98%identity with SEQ ID NO:2, or a fragment of a specified protein having cellulase activity.

5. Fused protein according to claim 1, where endoglucanase core has at least 99%identity with SEQ ID NO:2, or a fragment of a specified protein having cellulase activity.

6. Fused protein according to claim 4, where endoglucanases the core contains amino acids 19-334 SEQ ID NO:2.

7. Fused protein according to claim 1, where the linker and CBD come from cellobiohydrolase Chaetomium thermophilum.

8. Fused protein according to claim 7, where the linker and CBD come from cellobiohydrolase S. thermophilum CBS 730.95.

9. Fused protein according to claim 1, where endoglucanases the core contains the sequence of SEQ ID NO:2, and the linker and CBD contain the sequence of SEQ ID NO:15.

10. Fused protein according to claim 1, where endoglucanases the core genome is encoded, which is included in the strain E. coli DSM 17326.

11. Fused protein according to claim 1, which is encoded fused gene, which is included in the strain E. coli DSM 18159.

12. Selected polynucleotide encoding cellulase protein and is selected from the group including:
a) the nucleotide sequence of SEQ ID NO:3;
b) the sequence obtained in accordance with the degeneracy of the genetic code, on the basis of sequence (a) and
C) a nucleotide sequence encoding a fusion protein containing the first amino acid serial is inost endoglucanases kernel having at least 95%identity with SEQ ID NO:2 and cellulase activity, or a fragment of a specified protein having cellulase activity and a second amino acid sequence containing the linker and cellulose-binding domain (CBD)that has at least 95%identity with SEQ ID NO:15 and cellulose-binding activity, or a fragment of a specified protein with cellulose-binding activity.

13. Polynucleotide indicated in paragraph 12, which represents a fused gene encoding cellulase protein according to claim 1.

14. The cellulase expression vector fused protein according to claim 1, bearing polynucleotide on item 12.

15. A host cell belonging to filamentous fungi, containing the expression vector for 14 and producing a fused protein according to claim 1.

16. A host cell according to item 15, referring to the species of fungi of the genus Trichoderma, containing expressive vector on 14 and producing a fused protein according to claim 1.

17. A method of obtaining a fused protein according to any one of claims 1 to 11, comprising transforming the host cell expressing the vector coding for this protein fusions, and the cultivation of the specified host cell under conditions enabling expression of the indicated protein fusions, and if necessary, the isolation and purification of protein produced by the cell merge.

18. An enzyme having cellulase and cellulose-binding activity and steradily protein according to any one of claims 1 to 11, obtained by the method comprising transformation of a host cell expressing a vector encoding the indicated protein, and culturing the specified host cell under conditions enabling expression of the specified fused protein, and optionally the isolation and purification of protein produced by the cell merge.

19. The method of processing cellulosic material, providing contacting cellulosic material with a fused protein according to any one of claims 1 to 11 or an enzyme preparation according p.

20. The method according to claim 19, where the cellulosic material is a textile material, a plant, used in animal feeding, or derived from wood pulp mass, or recycled fiber.

21. The way biological abrasive finishing of textiles (biostoning), including the state security contact cellulase fusion protein according to any one of claims 1 to 11 or an enzyme preparation according p with fabric denim or clothing from denim.

22. The way the final biological treatment of fabrics (biofinishing), including the state security contact cellulase fusion protein according to any one of claims 1 to 11 or an enzyme preparation according p with textile materials, such as cloth, or clothing, or thread.

23. Detergent composition containing the fusion protein according to any one of claims 1 to 11 and add detergent and for processing cellulosic material, such as textile material.

24. Animal feed containing the fusion protein according to any one of claims 1 to 11 to improve digestion and extent of digestibility of feed containing cellulosic material such as plant material.

26. The Escherichia coli strain containing the gene encoding a protein according to claim 1, deposited under number DSM 18159.



 

Same patents:

FIELD: medicine.

SUBSTANCE: strain is prepared of a commercial producer, a mutant strain, Aspergillus awamori M-2002 (Russian National Collection of Microorganisms F-3771D) with using methods of induced mutagenesis and DNA technologies. The strain A.awamori Xyl T-15 is deposited in Russian National Collection of Microorganisms of Scryabin Institute of Biochemistry and Physiology of Microorgnisms of Microorgnisms of the Russian Academy of Sciences No. 4278D, stored in a lyophilised condition on a mowed wort agar in the Department of Enzymatic Preparation in Food Industry of State Scientific Institution All-Russian Research Institution of Food Biotechnology of Moscow Russian Agricultural Academy.

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3 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention represents novel endo-(1-4)-β-D-xylanases of mycelial fungus Penicillium canescens. Invention also related to method of obtaining endo-(1-4)-β-D-xylanases with application of eukaryotic cells, transformed by fragment of DNA, coding said xylanases from Penicillium canescens.

EFFECT: invention makes it possible to extend arsenal of known endoxylanases.

12 cl, 6 dwg, 4 tbl, 7 ex

FIELD: medicine.

SUBSTANCE: proposed means to enhance cellulase activity is a β-(3',5'-ditret-butyl-4'-hydroxyphenyl) propionic acid (phenozan-acid) or its potassium salt (phenozan-K). The means shows the activating effect in a wide range of concentrations, including ultra-low.

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1 tbl, 1 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology and specifically to microbiological industry. Use of the given Aspergillus foetidus 379-K strain enables to obtain a complex of pectolytic enzymes with high level of activity of endopolygalacturonase, β-glucanase, xylanase, cellulose, mannanase and chitinase. The strain is obtained through multi-step selection from the Aspergillus foetidus 37(TMPM-P-270) strain using mutagenesis. The strain is stored in lyophilic dried state and on splayed wort agar.

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2 tbl, 3 ex

FIELD: biotechnologies.

SUBSTANCE: food carboxymethylcellulose is dissolved in buffer solution or alkalised distilled water with pH of 9.5-10.0, and cellulose produced by culture Trichoderma reesei or culture Trichoderma viride is added to filtrate of produced solution with activity of at least 50 units/g in the form of powder in the presence of 1-1.5% solution of surfactant in process of intense mixing with speed of 4500-5000 rpm. Produced emulsion is cooled down to 5-10°C, pH of reaction mixture is reduced down to 3.0-3.5 and is left for a day in refrigerator. Precipitated particles are separated from solution, and acetate buffer is added repeatedly to remained particles with pH of 4-4.5 in process of mixing, then precipitated particles that contain cellulase are separated and dried.

EFFECT: reduced dimensions of immobilised cellulase particles, increased speed of fermentative process.

3 ex

FIELD: medicine; microbiology.

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2 cl, 1 tbl, 3 ex

FIELD: medicine; pharmacology.

SUBSTANCE: strain of Myceliophthora fergusii UV-64 VKM F-3932D possesses ability to produce the complex of highly active carbohydrases including neutral cellulase, xylanase and beta-glucanase. It allows receiving variety of enzymes for hydrolysis of non-starched polysaccharides of the vegetative raw materials showing high activity at acescent and neutral value pH.

EFFECT: expansion of assortment of fermental preparations.

3 tbl, 6 ex

FIELD: production methods.

SUBSTANCE: stamp of bacterium Bacillus macerans ARC V-2419 D - producer of pectaltliase, pectilase and polygalactturonase and complex of alkaline carbohydrase, contains csilanse, β-glucanese, galactanese, arabinase and amylase can be used at micro-biological, food, textile, paper production, and also at food additions.

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2 ex

FIELD: biotechnology, biochemistry, enzymology.

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3 cl, 4 ex

FIELD: biotechnology, biochemistry, enzymes.

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EFFECT: valuable biochemical properties of enzyme.

17 cl, 1 ex

FIELD: chemistry.

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25 cl, 16 dwg, 7 tbl, 12 ex

FIELD: medicine.

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30 cl, 6 ex, 32 dwg, 39 tbl

FIELD: medicine.

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18 cl, 21 dwg, 4 tbl, 2 ex

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SUBSTANCE: compound contains IL-15 indirectly bound by covalent links with a polypeptide containing a sushi domain of an extra-cellular region of the alpha IL-15R subunit.

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44 cl, 42 dwg, 4 tbl, 1 ex

FIELD: medicine.

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6 cl, 15 dwg, 1 tbl, 13 ex

FIELD: medicine.

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18 cl, 2 ex, 10 dwg

FIELD: medicine, pharmaceutics.

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34 cl, 31 dwg, 5 tbl, 19 ex

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24 cl, 6 dwg, 12 ex

FIELD: chemistry.

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38 cl, 14 dwg, 25 tbl, 16 ex

FIELD: medicine.

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2 cl, 4 dwg, 2 ex

FIELD: medicine.

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44 cl, 42 dwg, 4 tbl, 1 ex

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