Method of treating cellulose material and enzymes used therein

FIELD: chemistry.

SUBSTANCE: cellobiohydrolase polypeptide is selected from a group consisting of: a polypeptide containing an amino acid sequence having at least 95% identity to SEQ ID NO:6 and having cellobiohydrolase activity and an fragment a), having cellobiohydrolase activity. Such polypeptides can be obtained using a recombinant technique using suitable polynucleotides, expression vectors and host cells.

EFFECT: invention can be used in production of fermentable sugars and bioethanol from lignocellulose material via enzymatic conversion.

32 cl,15 dwg, 31 tbl, 32 ex

 

The scope of the invention

The present invention relates to the production of hydrolyzed sugars from cellulosic material. More specifically the invention relates to the production of formatiruem sugars from lignocellulosic material by enzymatic conversion. Formatiruem sugar is used, for example, upon receipt of bioethanol or for other purposes. In particular the invention is directed to a method of processing cellulosic material with cellobiohydrolase, endoglucanase, beta-glucosidase and, if necessary, xylanase, and enzyme preparations and their applications. The invention is also directed to new cellulolyticus polypeptides, encoding their polynucleotides and vectors and host cells containing polynucleotide. In addition, the invention is directed to the use of polypeptides and the way they are received.

Prior art

The hydrolyzate sugars can be used for microbial getting multiple chemical compounds or biopolymers, such as organic acids, e.g. lactic acid or ethanol, and other alcohols, for example, n-butanol, 1,3-propane diol, or polyhydroxyalkanoates (PHAs). The hydrolyzate sugars can also serve as raw materials for other nemikrobnoy ways, for example for the enrichment, isolation and purification of Vysokomolekulyarnye what's sugars or different methods of polymerization. One of the main applications of the hydrolyzate sugars is the production of biofuel. The production of bioethanol and/or other chemical compounds may occur in the integrated process for bioremediation (Wyman, 2001).

Limited resources of fossil fuels and the increased number of released from CO2that causes the greenhouse effect, increased the need for the use of biomass as a renewable and clean source of energy. One of the promising alternative technologies is the production of biofuels, i.e. ethanol from cellulosic materials. In the transport sector currently, the biofuel is the only choice that could reduce the yield of CO2through regulation of its value. Ethanol can be used in existing vehicles and distribution systems, and therefore do not require expensive investments in infrastructure. Sugar derived from renewable lignocellulosic raw materials, can also be used as raw material for many chemical products that can replace chemical products oil-based.

Most of the hydrocarbons in plants are in the form of lignocellulose, which consists mainly of cellulose, hemicellulose, pectin and lignin. In the process of lignocellulose-ethanol of lign the cellulosic material is pretreated or physical, or chemical methods to make the cellulose fraction more accessible to hydrolysis. After this, the pulp fraction hydrolyzing to obtain sugar, which with the help of yeast can ferment into ethanol. As the main petroleum product get lignin, which can be used as a solid fuel.

The cost of ethanol production is high, and the output power is low, so constantly ongoing effort to make the process more economical. Enzymatic hydrolysis is considered as the most promising technology for the conversion of cellulosic biomass in formatiruem sugar. However, on an industrial scale hydrolysis of limited use, and especially this technology is unsatisfactory when used heavily significandy material, such as wood waste or agriculture. The cost of the enzymatic stage is one of the main economic factors of the process. Was made attempts to improve the efficiency of enzymatic hydrolysis of cellulosic material (Badger, 2002).

In the US 2002/0192774 A1 describes a continuous process for conversion of the solid lignocellulosic biomass into combustible fuel products. After pre-treatment by wet oxidation or rupture steam biomass is partially divided into a whole is olosu, the hemicellulose and lignin, and then subjected to partial hydrolysis with one or more carbohydrase enzymes (EC 3.2). In the example of a commercial product Celluclast™ company Novo Nordisk A/S, with cellulase and xylanase activities.

In the US 2004/000 5674 A1 describes a new mixture of enzymes that can be used directly on the lignocellulosic substrate, it is possible to avoid getting toxic waste generated during pre-processing, and can save energy. Synergistic blend of enzymes contains a cellulase and an additional enzyme, such as cellulase, xylanase, xylanase, ligninase, amylase, protease, lipids or glucuronidase, or any combination. It is understood that the cellulase comprises endoglucanase (EG), beta-glucosidase (BG) and cellobiohydrolase (CBH). The examples illustrate the use of a mixture of products of xylanase and cellulase Trichoderma.

Kurabi et al. (2005) investigated the enzymatic hydrolysis broken steam and pre-treated organic ethanol solvent of douglasii using new and commercial fungal cellulases. They tested two commercial cellulase preparation Trichoderma reesei and two new drug produced by mutant strains of Trichoderma sp. and Penicillum sp. Preparation from Trichoderma sp. showed significantly better ha is acteristic, than other drugs. It was assumed that the best features, at least partially, due to a significantly higher beta-glucosidase activity, which weakens the products that inhibit the action of cellobiohydrolase and endoglucanase.

US 2004/005 3373 A1 relates to a method for conversion of cellulose into glucose by treating pre-prepared lignocellulosic substrate with a mixture of enzymes, including cellulase and modified cellobiohydrolase I (CBHI). CBHI modified by inactivation of its pulp-binding domain (CBD). The advantages of the modification of CBHI are, for example, in the best recovery and a higher rate of hydrolysis at high substrate concentration. The cellulase selected from the group consisting of EG, CBH and BG. CBHI preferably derived from Trichoderma.

In the US 2005/0164355 A1 describes a method for degradation of lignocellulosic material with one or more cellulolyticus enzymes in the presence of at least one sufactant. Can be used as auxiliary enzymes such as hemicellulase, esterase, peroxidase, protease, laccase, or a mixture thereof. The presence of sufactant enhances the degradation lignocellulose material compared to the absence of sufactant. Cellulolyticus the enzyme can be any enzyme that is included in degradati lignocellulose, including CBH, EG and BG.

There are a large number of publications, addressing various cellulase and hemicellulase.

For example, in WO 03/000 941 reveal cellobiohydrolases (CBHs), which belong to the CBHI-enzymes derived from fungi. Not provide any physiological properties of these enzymes, no examples of their application. Hong et al. (2003b) describe CBHI from Thermoascus aurantiacus, produced in yeast. Use of the enzyme has not been described. Tuohy et al. (2002) describe three forms of cellobiohydrolase from Talaromyces emersonii.

Endoglucanase family cel5 (EGs fam 5) describes, for example, in WO 03/062 409, which relates to compositions comprising at least two thermostable enzyme to use in food industries. Hong et al. (2003a) describe the production of a thermostable endo-β-1,4-glucanase from T.aurantiacus in yeast. Any applications not specified. WO 01/70998 refers to β-glucanase from Talaromyces emersonii. We discuss applications for the production of food, feed, beverage, brewing and detergents. Lignocellulosic hydrolysis is not mentioned. In WO 98/06 858 describes beta-1,4-endoglucanase from Aspergillus niger and discusses the application of this enzyme in the production of food and feed. WO 97/13853 describes methods for screening DNA fragments encoding enzymes in cDNA libraries. cDNA library has a yeast or fungal the origin is of preferably from Aspergillus. The enzyme is preferably a cellulase. Van Petegen et al. (2002) describe the 3D structure of endoglucanase cel5 family from Thermoascus aurantiacus. Parry et al. (2002) describe the principle of endoglucanase cel5 family from Thermoascus aurantiacus.

Endoglucanase family cel7 (EGs fam 7) are disclosed, for example, in US 5,912,157, which refers to the endoglucanases Myceliphthora and its homologues and their applications in the production of detergents, textile and pulp industry. US 6,071,735 describes cellulase detecting high endoglucanase activity in an alkaline environment. We discuss applications as detergent, pulp and paper and textile industries. Bioethanol is not mentioned. US 5,763,254 reveals the enzymes degrading cellulose/hemicellulose and having conservative amino acid residues in the CBD.

Endoglucanase family cel45 (EGs fam 45) are disclosed, for example, in US 6,001,639, which relates to enzymes having endoglucanase activity and containing two konservativnye amino acid sequence. Discussed in General use for the textile industry, as a detergent in the pulp and paper industry, processing of lignocellulosic material is mentioned, but no examples are not given. WO 2004/053039 aimed at application of endoglucanases as detergent. In the US 5,958,082 opens the application and the glucanase, especially from Thielavia terrestris, in the textile industry. EP 0495258 relates to detergent compositions containing cellulase Humicola. In the US 5,948,672 describes the preparation of cellulase containing endoglucanase, especially from Humicola and its application in textile and pulp industry. Hydrolysis of lignocellulose not mentioned.

A small amount of beta-glucosidase (BG) enhances the hydrolysis of biomass to glucose by splitting cellobiose produced by cellobiohydrolase. Conversion of cellobiose into glucose is usually the main limiting speed stage. Beta-glucosidase disclosed, for example, in US 2005/021 4920, which refers to BG from Aspergiilus fumigatus. This enzyme is produced in Aspergiilus oryzae and Trichoderma reesei. In General discusses the application of the enzyme in the degradation of biomass and as a detergent, but the examples are not illustrated. In WO 02/095 014 describes the enzyme from Aspergiilus oryzae having cellobiase activity. Use in the production of ethanol from biomass in General is discussed, but examples are not given. WO 2005/074656 discloses polypeptides having increased cellulolyticus activity taking place, for example, of the T. aurantiacus; A. fumigatus; T. terrestris and T. aurantiacus. In WO 02/26879 disclosed enzymatic processing of plant material. In the US 6,022,725 disclosed cloning and amplification of beta-glucosidase gene of Trichoderma reesei, the US 6,103,464 describes how the discovery of DNA, encodes a beta-glucosidase from a filamentous fungus. No examples of the application is not given.

Xylanase described, for example, in FR2786784, which refers to thermostable xylanase, useful, for example, in the processing of animal feed and bakery. The enzyme comes from fibrous fungus, in particular of the genus Thermoascus.

In the US 6,197,564 describes enzymes having xylanase activity derived from Aspergillus aculeatus. Illustrated by their use in baking. WO 02/24926 refers to the xylanase from Talaromyces. Examples of their use in food production and bakery. In WO 01/42433 disclosed thermostable xylanase from Talaromyces emersonii for use in food and feed production.

The most researched and widely used cellulolyticus enzymes of fungal origin come from Trichoderma reesei (anamorph of Hypocrea jecorina). Accordingly, the majority of commercially available fungal cellulases originated from Trichoderma reesei. However, most of the cellulases of the lesser-known fungi have not yet been practically important processes, such as degradation of cellulosic material, including lignocellulose.

There is a constant need for new methods of degradation of cellulosic substrates, in particular lignocellulosic substrates, and new enzymes and mixtures of enzymes, which is increasing the efficiency of degradation. There is also a need for methods and enzymes that work at high temperatures, which enables the use of biomass with a high density and leads to high concentrations of sugars and ethanol. This approach can lead to significant savings in energy and investment costs. High temperature, in addition, reduces the risk of infection during hydrolysis. The purpose of the present invention is to meet at least some of these needs.

Brief description of the invention

It's amazing, but it was found that cellulolyticus enzymes, and especially cellobiohydrolase derived from Thermoascus aurantiacus, Acremonium thermophilum, or Chaetomium thermophilum, extremely useful in the hydrolysis of cellulosic material. In addition cellobiohydrolase these fungi also contain endoglucanase, beta-glucosidase, and xylanase, which are very suitable for the degradation of cellulosic material. These enzymes are kinetically very effective in a wide temperature range, and although they have high activity at high temperatures, they are also very effective at standard temperature hydrolysis. This makes them extremely well suited for various processes of hydrolysis of cellulosic substrates by the traditional temperature and at elevated temperatures.

This is the invention suggests a method of treatment of cellulosic material with cellobiohydrolase, endoglucanase and beta-glucosidase, while mentioned cellobiohydrolase includes an amino acid sequence having at least 80%identity to SEQ ID NO: 2, 4, 6, or 8, or enzymatically active fragment.

The invention further provides an enzyme preparation comprising cellobiohydrolase, endoglucanase and beta-glucosidase, where the mentioned cellobiohydrolase includes an amino acid sequence having at least 80%identity to SEQ ID NO: 2, 4, 6, or 8, or enzymatically active fragment.

It is also proposed the application of the mentioned enzyme preparation for the degradation of cellulosic material, as well as the application of the mentioned method in the process of producing ethanol from cellulosic material.

The invention is also directed to a polypeptide comprising a fragment having cellulolyticus activity selected from the group consisting of:

a) a polypeptide comprising amino acid sequence having at least 66%identity to SEQ ID NO:4, 79%identity to SEQ ID NO:6, 78%identity to SEQ ID NO:12, 68%identity to SEQ ID NO:14, 72%identity to SEQ ID NO:16, 68%identity to SEQ ID NO:20, 74%identity to SEQ ID NO:22 or 24, or 78%identity to SEQ ID NO:26;

b) option (a), comprising a fragment having cellulolyticus activity; and

(C) fragment of a) or b), and housego cellulolyticus activity.

Another object of the invention is selected polynucleotide selected from the group consisting of:

a) the nucleotide sequence of SEQ ID NO: 3, 5, 11, 13, 15, 19, 21, 23 or 25, or a sequence that encodes a polypeptide according p claims;

b) thread complementary to a);

c) fragment of a) or b)comprising at least 20 nucleotides; and

d) a sequence that is degenerate as a result of the genetic code, in respect of any of the sequences presented in a), b) or c).

Further, the invention also provides a vector which comprises the above-mentioned polynucleotide as heterologous sequence and a host cell comprising the aforementioned vector. Strains of Escherichia coli having access number DSM 16728, DSM 16729, DSM 17324, DSM 17323, DSM 17729, DSM 16726, DSM 16725, DSM 17325 or DSM 17667, also included in the invention.

Other objectives of the invention are enzyme preparations comprising at least new polypeptides, and the use of these polypeptides or enzymatic products in the fuel, textile, pulp and paper, food industry, in the manufacture of detergents, feeds and beverages.

The following is a suggested method of obtaining a polypeptide comprising a fragment having cellulolyticus activity, the polypeptide selected from the group consisting of:

a) a polypeptide comprising AMI is kislotno sequence, having at least 66%identity to SEQ ID NO:4, 79%identity to SEQ ID NO:6, 78%identity to SEQ ID NO:12, 68%identity to SEQ ID NO:14, 72%identity to SEQ ID NO:16, 68%identity to SEQ ID NO:20, 74%identity to SEQ ID NO:22 or 24, or 78%identity to SEQ ID NO:26;

b) option (a), comprising a fragment having cellulolyticus activity; and

c) fragment of a) or b)having cellulolyticus activity; and

moreover, the method includes transforming the host cell with the vector coding for the said polypeptide, and culturing the aforementioned host cell under conditions enabling expression of the polypeptide, and, optionally, separation and purification of the resulting polypeptide.

it is also proposed a method of processing cellulosic material with the spent culture medium of at least one microorganism capable of producing the polypeptide, it is described above, where the method includes reacting the cellulosic material with waste environment cultivation to obtain hydrolyzed cellulose material.

Variants of embodiments of the invention are presented in the dependent claims.

Other objectives, features and advantages of the present invention will become apparent from the subsequent drawings, detailed description and examples.

A brief description of the drawing the

Figure 1. The temperature dependence of cellulase and beta-glucosidase activities within supernatant six tested fungal strains. The incubation time in this analysis was 60 min at this temperature, the analysis of pH was 5.0 (MUL-activity) or 4.8 (CMCase or BGU). The activity obtained at 60aboutWith presented as a relative activity of 100%. A) Thermoascus aurantiacus ALKO4239, B) Thermoascus aurantiacus ALKO4242, C) Acremonium thermophilum ALKO4245, D) Talaromyces thermophilus ALKO4246, E) Chaetomium thermophilum ALKO4261, F) Chaetomium thermophilum ALKO4265.

Figure 2. Schematic representation of expression cassettes used to transform protoplasts of Trichoderma reecei for producing recombinant fungal proteins. Recombinant genes were under the control of T. reecei cbh1 (cel7A) promoter (cbh1 prom), and a transcription termination is guaranteed by the use of a termination sequence T. reecei cbh1 (cbh1-term). As a marker of transformation was included amdS gene.

Figure 3. A) pH-Optima of the recombinant protein CBH/Cel7 from Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245 defined by 4-methylumbelliferyl-β-D-lactoside (MUL) at 50aboutWith in 10 minutes the Results are shown as average (±SD) of three separate measurements. B) Thermal stability of recombinant protein CBH/Cel7 from Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245 defined by 4-meta umbelliferyl-β-D-lactoside (MUL) at the optimum pH for 10 minutes The results are shown as average (±SD) of three separate measurements. Both reactions contained BSA (100 μg/ml) as a stabilizer.

Figure 4. The hydrolysis of crystalline cellulose (Avicel) using purified recombinant cellobiohydrolase at 45aboutC. the substrate Concentration 1% (wt/V), pH 5.0, the concentration of enzyme of 1.4 μm. A) cellobiohydrolase containing CBD, B) cellobiohydrolase (cor) without CBD.

Figure 5. The hydrolysis of crystalline cellulose (Avicel) using purified recombinant cellobiohydrolase at 70aboutC. the substrate Concentration 1% (wt/V), pH 5.0, the concentration of enzyme of 1.4 μm. A) cellobiohydrolase containing CBD, B) cellobiohydrolase (cor) without CBD.

Figure 6. A) pH dependence of the activity heterologic produced by Acremonium EG_40/Cel45A, EG_40_/Cel45B and Thermoascus EG_28/Cel5A was determined using the CMC substrate within a 10-minute reaction at 50aboutC. B) Temperature optimum Acremonium EG_40/Cel45A, EG_40_/Cel45B and Thermoascus EG_28/Cel5A was determined at pH 5.5, 4.8 and 6.0, respectively. The reaction involving the CMC as substrate was carried out for 60 min, except EG_28/Cel5A, which was carried out for 10 minutes as a stabilizer was added BSA (100 µg/ml).

Figure 7. A) pH dependence of the activity heterologic produced by Acremonium BG_101/Cel3A, Chaetomium BG_76/Cel3A, and Thermoascus BG_81/Cel3A was determined using 4-nitrophenyl-β-D-glucopyranoside substrate 10 minutes of reaction at 50 aboutC. B) Temperature optimum Acremonium EG_40/Cel45A, EG_40_ similar/Cel45B and Thermoascus EG_28/Cel5A was determined at pH 4.5, 5.5, and 4.5, respectively. Reaction with 4-nitrophenyl-β-D-glucopyranoside as substrate was carried out for 60 min, as a stabilizer was added BSA (100 µg/ml).

Figure 8. A) pH dependence of xylanase activity heterologic produced Thermoascus XYN_30/Xyn10A was determined using substrate from birch xylan within 10 minutes of reaction at 50aboutC. B) Temperature optimum XYN_30/Xyn10A was determined at pH 5.3 during the 60-minute reaction, as a stabilizer was added BSA (100 µg/ml).

Figure 9. Hydrolysis of washed torn ferry fiber eating (10 mg/ml) with a mixture of thermophilic enzymes (MIXTURE 1) and enzymes from T. reesei at 55 and at 60aboutC. the Dosage of enzymes is given in FPU units filter paper)/g of dry matter of the substrate, FPU analyzed 50aboutC, pH 5. The hydrolysis was carried out for 72 h at pH 5, with stirring. The results are given as mean (±SD) of three separate measurements.

Figure 10. Hydrolysis of washed torn steam corn straw (10 mg/ml) with a mixture of thermophilic enzymes (MIXTURE 2) and enzymes from T. reesei at 45, 55 and 57.5aboutC. the Dosage of enzymes for MIXTURE 2 was 5 FPU/g dry matter of substrate and enzymes from T. reesei - 5 FPU/g dry matter Celluclast, supplemented with 100 ncat/g dry in the society Novozym 188 (activity of filter paper was analyzed under 50 aboutC, pH 5). The hydrolysis was carried out for 72 h at pH 5, with stirring. The results are given as mean (±SD) of three separate measurements. The substrate contained soluble restored sugar (about 0.7 mg/ml). This background concentration of sugars was subtracted from the contents of the recovered sugars formed during hydrolysis.

Figure 11. Hydrolysis of washed torn steam corn straw (10 mg/ml) with a mixture of thermophilic enzymes containing a new thermophilic xylanase from Thermoascus aurantiacus (MIX 3) and enzymes from T. reesei at 45, 55 and 60aboutC. the Dosage of enzymes for MIXTURE 3 was 5 FPU/g dry matter of substrate and enzymes from T. reesei - 5 FPU/g dry matter Celluclast, supplemented with 100 ncat/g dry matter Novozym 188 (activity of filter paper was analyzed under 50aboutC, pH 5). The hydrolysis was carried out for 72 h at pH 5, with stirring. The results are given as mean (±SD) of three separate measurements. The substrate contained soluble restored sugar (about 0.7 mg/ml). This background concentration of sugars was subtracted from the contents of the recovered sugars formed during hydrolysis.

Figure 12. Hydrolysis of washed torn ferry fiber eating (10 mg/ml) with a mixture of thermophilic enzymes containing a new thermophilic xylanase XYN_30/Xyn10A from Thermoascus aurantiacus (MIX 3) and enzymes from T. reesei at 45, 55 and 60aboutC. Dosiro the CA enzyme to a MIXTURE of 3 was 5 FPU/g dry matter of the substrate, and for enzymes from T. reesei - 5 FPU/g dry matter Celluclast, supplemented with 100 ncat/g dry matter Novozym 188 (activity of filter paper was analyzed under 50aboutC, pH 5). The hydrolysis was carried out for 72 h at pH 5, with stirring. The results are given as mean (±SD) of three separate measurements.

Figure 13. The effect of glucose on the activity of different β-glucosidase drugs. Standard analysis using p-nitrophenyl-β-D-glucopyranoside as substrate were carried out in the presence of glucose in the analyzed mixture.

Figure 14. FPU activity of mixtures of enzymes at temperatures from 50 to 70aboutWith presented as the percentage of activity under standard conditions (50aboutS, 1 h).

Figure 15. Relative cellulase activity of two different strains of T. reesei grown in media containing raw Nutriose (N0) or BG_81/Cel3A, pretreated with Nutrioso (NBG81)as carbon source.

Detailed description of the invention

Cellulose is the main structural component of higher plants. It provides plant cells high bend strength, helping them to resist mechanical stress and osmotic pressure. Cellulose is a β-1,4-glucan, built of linear chains of glucose residues connected by β-1,4-glycosidic bonds. Cellobiose t is aetsa the smallest repeating unit of cellulose. In the cell walls of cellulose Packed in differently oriented layers, which are embedded in a matrix of hemicelluloses and lignin. Hemicellulose is a heterogeneous group of hydrocarbon polymers containing mainly various glucans, Kilani and mannans. Hemicellulose consists of a linear frame of the β-1,4-linked residues substituted with short side chains, usually containing acetyl, glucuronyl, arabinosyl and galactosyl. Hemicellulose can be chemically cross-linked to lignin. Lignin is a complex stitched cross links the polymer variously substituted p-hydroxyphenylpropionic units that provide power cell stacks to withstand mechanical stress, and protects the cellulose from enzymatic hydrolysis.

Lignocellulose is a combination of cellulose and hemicellulose and polymers of phenol-propanolol units and lignin. It is physically hard, dense and impervious, and is the most common biochemical material in the biosphere. Containing lignocellulose materials are: for example, hard and soft wood chips, pulp, sawdust, waste timber and woodworking industry; agricultural biomass, such as straw, grasses, vines, sugar beet, straw and corn cobs, the Ohm sugar cane, the stems, leaves, empty pods, husks and the like; solid waste, Newspapers and discarded office paper, waste from grinding, for example, grain; giving energy crops (e.g. willow, poplar, switchgrass, reed Canary grass and so on). Preferred examples are corn straw, millet straw of cereals, sugar cane and originating from wood materials.

"Cellulosic material"as that term is used here, refers to any material, including cellulose, hemicellulose and/or lignocellulose as an essential component. "Lignocellulosic material" means any material, including lignocellulose. Such materials are, for example, plant materials, such as wood, including soft wood and hard wood, grass, agricultural crops, crop residues, pulp and pieces of paper, paper waste, waste food and feed industry, etc. Textile fibres such as cotton, fiber derived from cotton, flax, hemp, jute and man-made fibers such as modal, viscose, Lyocell, are typical examples of cellulosic materials.

In nature, cellulose material are degraded a number of different organisms, including bacteria and fungi. In a typical case, the cellulose is degradiruete different cellulases, acting sequentially or simultaneously. For the biological conversion of cellulose into glucose usually requires three types of hydrolytic enzymes: (1) Endoglucanase that cut internal beta-1,4-glucoside communication; (2) Ectocervical which cut the disaccharide glycosides of cellobiose from the end cellobiose polymer chain; (3) Beta-1,4-glucosidase, which hydrolyzing cellobiose and other short cello-oligosaccharides into glucose. In other words, the three major groups of cellulases are cellobiohydrolase (CBH), endoglucanases (EG) and beta-glucosidase (BG).

For the degradation of more complex cellulosic substrates require a wide range of different enzymes. For example, lignocellulose are degraded by hemicellulase, as well as xylanase and manasama. Hemicellulase is an enzyme gidrolizuut the hemicellulose.

"Cellulolyticus enzymes" are enzymes that have "cellulolyticus activity", which means that they are able to hydrolyze cellulosic substrates or their derivatives into smaller sugars. Cellulolyticus enzymes, therefore, include both cellulase and hemicellulase. Cellulase, as the term is used here, include cellobiohydrolase, endoglucanase and beta-glucosidase.

T. reesei has a well-known and efficient cellulase system, with the containing a series of two CBH-basics, two major and several minor EG-I and BG-AZ. CBHI (Cel7A) T. reesei breaks down sugar from the reducing end of the cellulose chain, contains a C-terminal cellulose-binding domain (CBD) and can be up to 60% of the total secreted protein. CBHII (Cel6A) T. reesei breaks down sugar non-reducing end of the cellulose chain contains N-terminal cellulose-binding domain, and can be up to 20% of the total secreted protein. Endoglucanase EGI (Cel7B) and EGV (Cel45A) have CBD at its C-end, EGII (Cel5A) has CBD at N-end, and EGIII does not contain cellulose-binding domain. CBHI, CBHII, EGI and EGII are the so-called "main cellulases" Trichoderma, including together 80-90% of the total secreted protein. Specialists in the art it is known that the enzyme can be active on multiple substrates, and enzyme activity can be measured using various substrates, methods and conditions. Identification of various cellulolyticus activities is discussed, for example, van Tilbeurgh et al., 1988.

In addition to the catalytic domain/bark expressing cellulolyticus activity, cellulolyticus enzymes can include one or more binding domains (CBDs), also known as carbohydrate-binding domains and/or modules (CBD/CBM), which can be located either at the N-or C-end of the catalytic domain. CBDs have carbohydrate-with asiausa activity and mediates the binding of cellulase with crystalline cellulose, but have weak or ineffective cellulase activity of the enzyme on soluble substrates. There are two domains, typically connected through a flexible and vysokopotentsirovannye linker region.

"Cellobiohydrolase" or "CBH"as that term is used here, refers to enzymes that break down cellulose from the end of the glucose chain and produce mainly cellobiose. They are also called 1,4-beta-D-glucan by cellobiohydrolase or pulp-1,4-beta-cellobioside. They hydrolyzing 1,4-beta-D-glucoside connection with reducing or nereguliruemaia the end of the polymer containing the mentioned relationships, such as cellulose, thereby making cellobiose. Two different CBHs were vydeleny from Trichoderma reesei, CBHI and CBHII. They have a modular structure consisting of a catalytic domain, coupled with the pulp-binding domain (CBD). In nature there are also cellobiohydrolase in which CBD is missing.

"Endoglucanase" or "EG" refers to enzymes that cut the internal glycosidic bonds in the cellulose chain. They are classified as EC 3.2.1.4. They are 1,4-beta-D-glucan 4-glucanohydrolase and catalyze endohedrals 1,4-beta-D-glycosidic linkages in the glucose polymers such as cellulose and its derivatives. Some found in nature, endoglucanase have pulp and connecting to the Yong, while others do not have it. Some endoglucanase have also xylanase activity (Bailey et al., 1993).

"Beta-glucosidase" or "BG", or "βG" refers to enzymes that degrade poorly soluble oligosaccharides, including cellobiose, to glucose. They are classified as EC 3.2.1.21. They are beta-D-glucoside by glycohydrolase that a typical catalyze the hydrolysis of terminal nereguliruemyi residues in beta-D-glucose. These enzymes recognize oligosaccharides of glucose. Typical substrates are cellobiose and allotrios. Cellobiose is an inhibitor of cellobiohydrolase, for this reason, the degradation of cellobiose is important to overcome the end-product inhibition of cellobiohydrolase.

Xylanase is an enzyme that can recognize to hydrolyze the hemicellulose. They include ecohydraulics and andoperations enzymes. In the typical case they have endo-1,4-beta-xylanase (EC 3.2.1.8) or beta-D-xyloside (EC 3.2.1.37) activity, which breaks down the hemicellulose to xylose. "Xylanase" or "Xyn" in connection with the present invention relates in particular to the enzyme classified as EC 3.2.1.8, hydrolysis by xylose polymers lignocellulosic substrate or purified xylan.

In addition to the cellulase can be classified according to R the EIT glycosylglycerols families according to their primary sequence, confirmed by the analysis of three-dimensional structures of some family members (Henrissat 1991, Henrissat and Bairoch 1993, 1996). Some glycosylglycerols are multifunctional enzymes that contain catalytic domains belonging to different glycosylglycerols families. Family 3 consists of beta-glucosidase (EC 3.2.1.21), such as described here Ta BG_81, At BG_101 and Ct BG_76. Family of 5 (formerly known as celA) consists mainly of endoglucanases (EC 3.2.1.4), such as described here Ta EG_28. Family 7 (previously cellulase family celC) contains endoglucanase (EC 3.2.1.4) and cellobiohydrolase (EC 3.2.1.91), such as described here Ct EG_54, Ta CBH, At CBH_A, At CBH_C and Ct CBH. Family 10 (previously celF) consists mainly of xylanases (EC 3.2.1.8), such as described here Ta XYN_30 and At XYN_60. The family 45 (previously celK) contains endoglucanase (EC 3.2.1.4), such as described here At EG_40 and At EG_40_.

Cellulolyticus enzymes useful for the hydrolysis of cellulosic material that is derived from Thermoascus aurantiacus, Acremonium thermophilum, or Chaetomium thermophilum. "Derived from" means that they can be derived from these species, but this does not exclude the possibility of obtaining them from other sources. In other words, they can be from any organism, including plants. Preferably they originate from microorganisms, for example bacteria or fungi. Bacter is and can be, for example, of a type selected from Bacillus, Azospirillum and Streptomyces. More preferably they originate from fungi (including filament fungi and yeast), for example, of the genus selected from the group consisting of Thermoascus, Acremonium, Chaetomium, Achaetomium, Thielavia, Aspergillus, Botritis, Chrysosporium, Collibia, Fomes, Fusarium, Humicola, Hypocrea, Lentinus, Melanocarpus, Myceliophthora, Myriococcum, Neurospora, Penicillium, Phanerochaete, Phlebia, Pleurotus, Podospora, Polyporus, Rhizoctonia, Scytalidium, Pycnoporus, Trametes and Trichoderma.

In accordance with the preferred embodiment of the invention the enzymes are obtained from strain ALKO4242 Thermoascus aurantiacus, deposited as CBS 116239, strain ALKO4245, deposited as CBS 116240 and classified currently as Acremonium thermophilium, or strain ALKO4265 Chaetomium thermophilum, deposited as CBS 730.95.

Cellobiohydrolase preferably includes an amino acid sequence having at least 80%identity to SEQ ID NO: 2, 4, 6, or 8, or enzymatically active fragment.

2
CellobiohydrolaseGeneDerived fromCBDNucleic acid SEQID NO:Amino acid SEQ ID NO:
Ta CBHTa cel7AT. aurantiacus-1
At CBH_AAt cel7BA. thermophilum-34
At CBH_CAt cel7AA. thermophilum+56
Ct CBHCt cel7AC. thermophilum+78

These CBHs have a better constant pulp inhibition compared with the corresponding constant CBH Trichoderma reesei, and they show the best results in hydrolysis when testing different cellulosic substrates. SEQ ID NO: 2 and 4 do not include the CBD. Especially the increased hydrolysis results can be obtained when the pulp-binding domain (CBD) attached to CBH, which does not have its own CBD. CBD can be obtained, for example, species of Trichoderma or Chaetomium, and preferably it is attached to CBH by the linker. The resulting fused protein containing CBH-crustal region attached to the CBD via a linker, may include amino acid sequence having at least 80%identity to SEQ ID NO: 28 or 30. Such fused proteins is deruyts polynucleotide, including the sequence of SEQ ID NO: 27 or 29.

The endoglucanases may include amino acid sequence having at least 80%identity to SEQ ID NO: 10, 12, 14 or 16, or its enzymatically active fragment. These endoglucanase have good thermostability.

Beta-glucosidase may include amino acid sequence having at least 80%identity to SEQ ID NO: 22, 24 or 26, or permutative active fragment. These beta-glucosidase have good resistance to glucose inhibition, which advantageously avoids the inhibition of the final product during the enzymatic hydrolysis of cellulosic material. Beta-glucosidase can be used to obtain sophorose, cellulase inducer used in the cultivation of T. reesei.

The endoglucanasesGeneDerived fromCBDN. acid SEQ ID NO:Amino acid SEQ ID NO:
Ta EG_28Ta cel5AT. aurantiacus-910
At EG_40At cel45AA. thermophilum+1112
At EG40_.At cel45BA. thermophilum-1314
Ct EG_54Ct cel7BC. thermophilum+1516
BetaglucosilatedGeneDerived fromN. acid SEQ ID NO:Amino acid SEQ ID NO:
Ta BG_81Ta cel3AT. aurantiacus2122
At BG_101At cel3AA. thermophilum2324
Ct BG_76Ct cel3AC. thermophilum2526

Xylanase may include consecutive amino acid is required, having at least 80%identity to SEQ ID NO: 18 or 20, or permutative active fragment.

XylanaseGeneDerived fromCBDN. acid SEQ ID NO:Amino acid SEQ ID NO:
Xyn_30Ta xyn10AT. aurantiacus+1718
Xyn_60At xyn10AA. thermophilum-1920

The term "identity" here refers to the overall identity between two amino acid sequences that are compared with one another, from the first amino acid encoded by the corresponding gene to the last amino acid. The identities of the sequences of full length is measured using the General alignment Needleman-Wunsch software package EMBOSS (European Molecular Biology Open Software Suite; Rice et al., 2000), version 3.0.0, with the following parameters: EMBLOSU62, the penalty for a gap 10.0, extension penalty of 0.5. The algorithm described in Needleman and Wunsch (1970). Specialist in the art ovadal the n about the results of using the algorithm of Needleman-Wunsch, comparable only when aligned with a corresponding domain sequence. Therefore, comparison of, for example, cellulase sequences, including CBD or signal sequences, sequences which do not have these items, cannot be performed.

In accordance with one embodiment of the invention is used cellulolyticus polypeptide that has at least 80, 85, 90, 95 or 99%identity with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26, or enzymatically active fragment.

The term "enzymatically active fragment" means a fragment of a sequence that has cellulolyticus activity. In other words, the enzymatically active fragment may be part of the Mature protein sequence, or it may be only a fragment of the Mature protein, provided that he still has cellobiohydrolase, endoglucanase, beta-glucosidase or xylanase activity.

Cellulolyticus enzymes, preferably, are recombinant enzymes, which can be obtained by a commonly known method. Allocate a polynucleotide fragment containing the gene of the enzyme, the gene embed under the control of a strong promoter of the expression vector, the vector is introduced into appropriate host cell and cultivated these host cell under conditions conducive to the formation of the enzyme. A method of producing a protein using recombinant technology in various systems of the hosts are well known in the art (Sambrook et al., 1989; Coen, 2001; Gellissen, 2005). Preferably the enzymes are produced as extracellular enzymes, which are secreted into the culture medium from which it can be easily restored and highlighted. Can be used spent culture medium producing master as such, or the host cell can be extracted from the environment, and/or can be otkorektirovat, filtered and fractionate. It can also be dried.

The selected polypeptide in this context may simply mean that the cells and cell debris removed from the culture medium containing the polypeptide. It is convenient to distinguish polypeptides, for example, adding anionic and/or cationic polymers in an elaborate culture medium to enhance the precipitation of cell debris and some enzymes, which have unwanted side activity. The medium is then filtered using inorganic filter agent and the filter to remove the precipitate. After that, the filtrate is treated using a semipermeable membrane, for at the Alenia excess salts, sugars and metabolic products.

In accordance with one embodiment of the invention the heterologous polynucleotide includes gene, similar to that included in the microorganism having the access number DSM 16723, DSM 16728, DSM 16729, DSM 16727, DSM 17326, DSM 17324, DSM 17323, DSM 17729, DSM 16724, DSM 16726, DSM 16725, DSM 17325 or DSM 17667.

Producing cell can be any organism that is able to Express cellulolyticus enzyme. Preferably the host cell is a microbial cell, more preferably a fungus. More preferably the host is a filament fungus. Preferably, the recombinant host modified for expression and secretion of cellulolyticus enzymes as its main activity or one of the main activities. This can be done by dellarovere main homologous secreted genes, for example, four main Trichoderma cellulases and direction of heterologous genes in the locus, which is modified to provide a high level of expression and production. The preferred hosts for producing cellulolyticus enzymes are, in particular, strains of the genus Trichoderma or Aspergillus.

The enzymes required for hydrolysis of cellulosic material, in accordance with the invention can be added to fermentative effective amount or at the same time, voltage is emer, in the form of enzyme mixture, or sequentially, or as part of a simultaneous sacharification and fermentation (SSF). Any combination of cellobiohydrolase comprising amino acid sequence having at least 80%identity to SEQ ID NO: 2, 4, 6, or 8, or enzymatically active fragment, can be used in conjunction with any combination of endoglucanases and beta-glucosidase. If cellulosic material includes hemicellulose, hemicellulose, for degradation, preferably, in addition, use of xylanase. Endoglucanase, beta-glucosidase and xylanase can be selected from those described here, but are not limited to. They can also be, for example, commercially available preparations of enzymes. In addition to cellulases and, if necessary, hemicellulase, you can use one or more other enzymes, such as protease, amylase, lactase, lipase, pectinase, esterase and/or peroxidase. The processing of other enzymes can be performed before, during or after cellulase treatment.

The term "enzyme preparation" refers to compositions comprising at least one of the desired enzymes. The drug may contain enzymes in at least partially purified and dedicated manner. An alternative drug may be spent culture medium or filtrate containing the Dean or more cellulolyticus enzymes. In addition cellulolyticus activity of the drug may contain additives, such as mediators, stabilizers, buffers, preservatives, sufactant and/or components of the environment of the cultivation. "A proven environment cultivation" refers to the environment of the cultivation of the owner, including the produced enzymes. Preferably the host cell is separated from the environment after producing enzymes.

In accordance with one embodiment of the invention the enzyme preparation comprises a mixture of CBH, EG and BG, if necessary together with xylanase and/or other enzymes. CBH includes an amino acid sequence having at least 80%identity to SEQ ID NO: 2, 4, 6, or 8, or enzymatically active fragment, which can be derived from Thermoascus aurantiacus, Acremonium thermophilium or Chaetomium thermophilum, while EG, BG and xylanase may have any origin, including from the mentioned organisms. Other enzymes that could be represented in the product are, for example, protease, amylase, laccase, lipase, pectinase, esterase and/or peroxidase.

In order to meet various process conditions, can be used in different enzyme mixtures and combinations. For example, if the degradation process should be carried out at high temperature, are selected thermostable enzymes. Combines what I CBH family of 7 with endoglucanase family 45, if necessary, in combination with the BG family of 3 and/or xylanase family of 10 had excellent results hydrolysis as at 45aboutWith, and at elevated temperatures.

Cellulolyticus enzymes of Trichoderma reesei are traditionally used in the hydrolysis at temperatures in the range of about 40-50aboutAnd 30-40aboutIn SSF. CBH, EG, BG and Xyn derived from Thermoascus aurantiacus, Acremonium thermophilium or Chaetomium thermophilum, also effective at these temperatures, but in addition, most of them also function exceptionally well at temperatures between 50 and 70aboutWith or even up to 80 and 85aboutS, such as between 55 and 70aboutWith, for example, between 60 and 65aboutC. Enzyme mixture, inkubirovanie for short periods of time, functional even up to 85aboutS, while for complete hydrolysis of the commonly used lower temperatures.

The method of processing cellulosic material with CBH, EG, BG and Xyn is particularly suitable for producing formatiruem sugars from lignocellulosic material. Formatiruem sugar can be fermented by yeast into ethanol and used as fuel. They can also be used as intermediates or raw materials for production of various chemicals or building blocks in the chemical industry, for example, in the so-called bioremediation. Lignocellulose the first material may be pre-treated prior to enzymatic hydrolysis, to destroy the fibrous structure of the cellulose substrates and make the cellulose fraction more accessible to cellulolyticus enzymes. Currently known pre-treatment include mechanical, chemical or thermal processes and their combinations. For example, the material may be pre-treated steam rupture or acid hydrolysis.

In Thermoascus aurantiacus, Acremonium thermophilium and Chaetomium thermophilum was found a number of new cellulolyticus polypeptides. New polypeptides can include a fragment that has cellulolyticus activity, and can be selected from the group consisting of a polypeptide comprising the amino acid sequence having at least 66%, preferably 70%or 75%, identity to SEQ ID NO: 4, 79%identity to SEQ ID NO: 6, 78%identity to SEQ ID NO: 12, 68%, preferably 70%or 75%, identity to SEQ ID NO: 14, 72%, preferably 75%, identity to SEQ ID NO: 16, 68%, preferably 70%or 75%, identity to SEQ ID NO: 20, 74%identity to SEQ ID NO: 22 or 24, or 78%identity to SEQ ID NO: 26.

New polypeptides may also be variants of the above polypeptides. "Option" may be a polypeptide, which occurs under natural conditions, such as allelic variant within the same strain, species or genus, or he could be educated on what redstem mutagenesis. It can include amino acid replacements, deletions or insertions, but still works essentially the same way with enzymes, defined above, i.e. it includes the fragment with cellulolyticus activity.

Cellulolyticus the polypeptide generally are produced in the cell as a crude polypeptide comprising a signal sequence that is cleaved during the secretion of the protein. They also can be further processionary during secretion both at the N-end and/or at the C-end with the formation of Mature, enzymatically active protein. A polypeptide "fragment, having cellulolyticus activity", therefore, means that the polypeptide can be either immature or Mature form, preferably, it is in the Mature form, i.e. the processing took place.

New polypeptides can also be, as mentioned above, "a fragment of the polypeptides or variants". The fragment can be a Mature form of the above-mentioned proteins or he may be the only enzyme active part of the Mature protein. In accordance with one embodiment of the invention the polypeptide has an amino acid sequence having at least 80, 85, 90, 95 or 99%identity to SEQ ID NO: 4, 6, 12, 14, 16, 20, 22, 24 or 26, or cellulolyticus active fragment. He also mo is et to be a variant or a fragment, having cellobiohydrolase, endoglucanase, xylanase or beta-glucosidase activity. In accordance with another variant embodiment of the invention, the polypeptide consists of especially cellulolyticus active fragment of the sequence SEQ ID NO: 4, 6, 12, 14, 16, 20, 22, 24 or 26.

New polynucleotide may include the nucleotide sequence of SEQ ID NO: 3, 5, 11, 13, 15, 19, 21, 23 or 25, or a sequence encoding a new polypeptide, as defined above, including comlementary them threads. Polynucleotide, as the term is used here, refers to both RNA and DNA, and it can be single-stranded or donativum. Polynucleotide may also be a fragment of the above-mentioned polynucleotide comprising at least 20 nucleotides, for example at least 25, 30 or 40 nucleotides. In accordance with one embodiment of the invention it has length at least 100, 200 or 300 nucleotides. Further polynucleotide may be degenerate as a result of the genetic code with respect to any of the sequences as defined above. This means that the same amino acid can encode different codons.

In accordance with one embodiment of the invention polynucleotide is included in SEQ ID NO: 3, 5, 11, 13, 15, 19, 21, 23 or 25, which means that this sequence contains at least part of at Omanthai sequence. In accordance with another variant embodiment of the invention polynucleotide includes gene, similar to that included in the microorganism having the access number DSM 16728, DSM 16729, DSM 17324, DSM 17323, DSM 17729, DSM 16726, DSM 16725, DSM 17325, DSM 17667.

New proteins/polypeptides can be prepared as described above. New polynucleotide can be embedded into a vector that can Express the polypeptide encoded by the heterologous sequence, and the vector can be embedded in a host cell that can Express the above-mentioned polypeptide. The host cell preferably belongs to the genus Trichoderma or Aspergillus.

Heterologous gene that encodes a new polypeptides is introduced into a plasmid or strain of Escherichia coli that has the access number DSM 16728, DSM 16729, DSM 17324, DSM 17323, DSM 17729, DSM 16726, DSM 16725, DSM 17325, DSM 17667.

New enzymes can be components of the enzyme preparation. The enzyme preparation can include one or more novel polypeptides, and may be, for example, in the form of spent culture medium, powder, granules or liquid. In accordance with one embodiment of the invention it includes cellobiohydrolase, endoglucanase, beta-glucosidase and, if necessary, xylanase activity and/or other enzymatic activity. It may also include any conventional additives.

New enzymes can be approx the tive in any process, includes cellulolyticus enzymes, such as fuel, textile, detergent, pulp and paper, food, feed industry and beverage industry, and in particular during the hydrolysis of cellulosic material for the production of biofuels, including ethanol. In the pulp and paper industry they can be used for the modification of cellulose fibers, for example, in the processing of Kraft pulp, mechanical pulp or recycled paper.

The invention is illustrated by the following non-limiting scope of the invention examples. Also it should be clear that the variations of the embodiment of the invention, the above description and examples are for illustrative purposes and within the scope of the invention various changes and modifications.

Examples

Example 1. Screening of strains exhibiting cellulolyticus activity, and their cultivation for cleaning

About 25 strains of fungi from culture collections Roal Oy was tested on cellulolyticus activity, including beta-glucosidase. After a preliminary screening for further research were selected 6 strains. It was Thermoascus aurantiacus ALKO4239 and ALKO4242, Acremonium thermophilium ALKO4245, Talaromyces thermophilius ALKO4246 and Chaetomium thermophilum ALKO4261 and ALKO4265.

Strains ALKO4239, ALKO4242 and ALKO4246 were cultivated in shake flasks at the 42aboutWith those who tell 7 d in medium 3 x In, which contains g/litre: Solka Floc cellulose 18, Barda from spent grain 18, xylan threshed oats 9, CaCO32, flour from soybeans 4,5, (NH4)HPO44,5, wheat bran 3,0, KH2PO41,5, MgSO4·H2O 1,5, NaCl 0.5, the gum from the beans acacia 9,0, trace element solution #1 to 0.5, trace element solution #2 0,5 and protivovospalitel Struktol (Stow, OH, USA), 0.5 ml; pH was regulated to 6.5. Trace element solution #1 contains g/l: MnSO41,6, ZnSO4·7H2O 3,45 and CoCl2·6H2O 2,0; trace element solution #2 contains, g/l: FeSO4·7H2O 5,0 two drops of concentrated H2SO4.

Strain ALKO4261 were cultivated in shake flasks in medium 1×In, which contains one third of each of the components of the environment 3 (above), except that it has the same concentration of CaCO3NaCl and trace elements. The strain was cultured at 45aboutC for 7 days

Strain ALKO 4265 cultivated in shake flasks in the following medium (g/litre: Solka Floc cellulose 40, Pharmamedia™ (Traders Protein, Memphis, TN, USA) 10, soaked powder corn 5, (NH4)2SO45 and KH2PO415; pH was regulated to 6.5. The strain was cultured at 45aboutC for 7 days

After culturing cells and other solid education besieged by centrifugation and recovered supernatant. During kultivirovanii throu rahimahullah flasks was added protease inhibitors PMSF (phenylmethyl-sulphonylchloride) and pepstatin And 1 mm and 10 μg/ml, respectively. If drugs are not used immediately, they were stored in aliquot at -20aboutC.

To assess thermoactive enzymes were conducted analyses of drugs, cultivated in shake flasks at 50, 60, 65, 70 and 75aboutC for 1 h in the presence of 100 μg of bovine serum albumin (BSA) /ml as a stabilizer. Preliminary analyses were performed at 50 and 65aboutWith two different pH values (4,8/5.0 or 6.0)to find out what pH was more appropriate for the analysis of thermoactivated.

All supernatant shake flasks were analyzed for the following activity:

Cellobiohydrolase I-like activity ("CBHI") and endoglucanases I-like activity ("EGI"):

They were measured in 50 mm Na-acetate buffer with 0.5 mm MUL (4-methylumbelliferyl-beta-D-lactoside) as substrate. For inhibition of any confounding beta glucosidase activity was added glucose (100 mm). Liberated 4-methylumbelliferyl was measured at 370 nm. "CBHI and EGI"activity differed by measuring activity in the presence and absence of cellobiose (5 mm). Activity that is not inhibited by cellobiose is "EGI"activity, and the remaining MUL-activity is "CBHI-activity (van Tilbeurgh et al., 1988). The analysis was performed at pH 5.0 or 6.0 (see below).

Endoglucanase (SMACNA) activity:

Beta glucosidase (BGU) activity:

It was analyzed using 4-nitrophenyl-β-D-glucopyranoside (1 mm) in 50 mm citrate buffer, as described by Bailey and Nevalainen 1981. Liberated 4-NITROPHENOL was measured at 400 nm. The analysis was carried out at a pH of 4.8 or 6.0 (see below).

The relative activity of the enzymes shown in Figure 1. The relative activity was the establishment of activity at 60aboutWith as 100% (Figure 1). All strains were produced enzymes that had high activity at high temperatures (65-75aboutC).

Regarding the purification of proteins. ALKO4242 were grown in 2-liter bioreactor (Braun patch BIOSTAT® B, Braun, Melsungen, Germany) in the following medium (g/litre: Solka Floc cellulose 40, flour from soybeans 10, NH4NO35, KH2PO45, MgSO4·7H2O 0,5, CaCl2·H2O 0,05, trace element solution #1 to 0.5, trace element solution #2 to 0.5. Aeration was 1 vvm, with control antispyware with Struktol, while stirring 200-800 rpm and the temperature of the 47aboutC. Conducted two groups of experiments, one at pH 4,7±0,2 (NH3/H2SO4and another with an initial pH of 4.5. Time of cultivation was 7 days After culturing cells and other solid education deleted what centrifugational.

Strain ALKO4245 were grown in 2-liter bioreactor (Braun patch BIOSTAT® B, Braun, Melsungen, Germany) in the following medium (g/litre: Solka Floc cellulose 40, soaked powder corn 15, Barda from spent grain 5, xylan threshed oats 3, gum from acacia beans 3, (NH4)2SO45 and KH2PO45. The interval pH was 5,2±0,2 (NH3/ H2SO4), aeration 1 vvm at stirring 300-600 rpm control antispyware with Struktol and the temperature of the 47aboutC. Time of cultivation was 7 days After culturing cells and other solid education was removed by centrifugation.

For purification of the enzyme ALKO4261 were grown in a 10 l bioreactor (Braun patch BIOSTAT® B, Braun, Melsungen, Germany) in the following medium (g/litre: Solka Floc cellulose 30, Barda from spent grain 10, xylan threshed oats, CaCO32, flour from soybeans 10, wheat bran 3,0, (NH4)2SO45, KH2PO45, MgSO4·7H2O to 0.5, NaCl 0.5, and KNO30,3, trace element solution #1 to 0.5, trace element solution #2 to 0.5. The interval pH was 5,2±0,2 (NH3/ H2SO4), aeration 1 vvm at stirring 300-600 rpm control antispyware with Struktol and the temperature of the 42aboutC. Time of cultivation was 5 D. the Second group was grown under similar conditions except that Solka Floc was added to 40 g/l, and the spent grain to 15 g/L. su is inatant recovered by centrifugation and filtration through a Seitz-150 K and EK-filters (Pall SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany). Then the supernatant was concentrated approximately ten-fold using ultrafiltration system Pellicon mini (NMWL filter 10 kDa; Millipore, Billerica, MA, USA).

For purification of the enzyme ALKO4265 also were grown in 10 l bioreactor (Braun patch BIOSTAT® B, Braun, Melsungen, Germany) in the same environment as mentioned above, except that KH2PO4added to 2.5 g/L. pH Interval was 5.3±0,3 (NH3/ H2SO4), aeration of 0.6 vvm while stirring at 500 rpm with control antispyware with Struktol and the temperature of the 43aboutC. Time of cultivation was 7 days the Supernatant was recovered by centrifugation and filtration through a Seitz-150 K and EK-filters (Pall SeitzSchenk Filtersystems GmbH, Bad Kreuznach, Germany). Then the supernatant was concentrated approximately twenty times using ultrafiltration system Pellicon mini (NMWL filter 10 kDa; Millipore, Billerica, MA, USA).

Example 2. Purification and characterization of cellobiohydrolase from Acremonium thermophilium ALKO4245 and Chaetomium thermophilum ALKO4265

Acremonium thermophilium ALKO4245 and Chaetomium thermophilum ALKO4265 were grown as described in Example 1. The main cellobiohydrolase was purified using based on p-aminobenzyl 1-thio-β-cellobioside affinity column, prepared as described Tomme et al., 1988.

Supernatant cultures were first butaritari in 50 mm sodium acetate buffer PH 5.0, containing 1 mm δ-gluconolactone and 0.1 M glucose that is edlite ligand hydrolysis in the presence of β-glucosidase. Cellobiohydrolase was suirable 0.1 M lactose and finally purified by gel-filtration chromatography using a column of Superdex 200 HR 10/30 in the ÄKTA system (Amersham Pharmacia Biotech). When gel filtration was used sodium phosphate buffer pH 7.0, containing 0.15 mm sodium chloride.

Peeled cellobiohydrolase were analyzed by electrophoresis on SDS-polyacrylamide gel and determined the molecular weight of both protein of approximately 70 kDa, estimated on the basis of molecular mass standards (Set for calibration of low molecular weights, Amersham Biosciences). Peeled cellobiohydrolase Acremonium and Chaetomium identified as At Cel7A and Ct Cel7A, respectively, following the scheme shown in Henrissat et al. (1998) (Henrissat, 1991; Henrissat and Bairoch, 1993).

The specific activity of the preparations was determined using as substrate 4-methylumbelliferyl-β-D-lactoside (MUL), 4-methylumbelliferyl-β-D-cellobioside (MUG2) or 4-methylumbelliferyl-β-D-tellurite (MUG3) (van Tilbeurgh et al., 1988) in 0.05 M sodium citrate buffer pH 5 at 50aboutC for 10 minutes Endoglucanase and xylanase activity was measured according to standard procedures (in accordance with IUPAC, 1987), using carboxymethyl cellulose (CMC) and glucuronosyl birch (Bailey et al., 1992) as substrates. Specific activity against Avicel was calculated on the basis of reducing sugars formed during the 24-hour R the shares at the 50 aboutC, pH 5.0, with 1% of the substrate and a dose of 0.25 μm enzyme. Protein in purified preparations of the enzyme was measured according to Lowry et al., 1951. In order to characterize the end-products of the hydrolysis of dissolved sugar released in a 24-hour hydrolysis experiment, as described above, were analyzed by HPLC (Dionex). Clean cellobiohydrolase I (CBHI/Cel7A from Trichoderma reesei was used as control.

The specific activity of purified enzymes and enzyme CBHI/Cel7A of T. reesei are presented in Table 1. Peeled cellobiohydrolase At Cel7A and Ct Cel7A have a higher specific activity against small synthetic substrates compared with CBHI/Cel7A of T. reesei. Specific activity against Avicel have disclosed here enzymes was clearly higher. Low activity of preparations of purified enzymes against xylan and CMC, probably due either to the properties of these proteins, or at least part of the remaining small amounts of contaminating enzymes. The main end product of cellulose hydrolysis using all of the purified enzymes was cellobiose, which is typical for cellobiohydrolase.

Table 1
Specific activity (ncat/mg) treated cellobiose Olas and control enzyme from T. reesei (50aboutC, pH 5.0, 24 h)
SubstrateA. thermophilum ALKO4245 Cel7AC. thermophilum ALKO4265 Cel7AT. reesei Cel7A
Xylan11,36,71,3
CMC26,25,51,0
MUG29,218,94,3
MUG31,31,50,9
MULa 21.554,0of 21.9
Avicel1,81,40,6

Thermal stability of purified cellobiohydrolase was measured at different temperatures. The reaction was carried out in the presence of 0.1% BSA at pH 5.0 for 60 min, using as substrate 4-methylumbelliferyl-β-D-lactoside. C. thermophilum ALKO4265 CBH/Cel7A and A. thermophilum ALKO4245 CBH/Cel7A were stable up to 50 and 60aboutC, respectively. The control enzyme from T. reesei (CBH/Cel7A) retained 100%activity is about 55 aboutC.

Example 3. Purification and characterization of endoglucanase from Acremonium thermophilum ALKO4245

Acremonium thermophilum ALKO4245 were grown as described in Example 1. The supernatant of the culture was incubated at 70aboutC for 24 hours, after which it was concentrated by ultrafiltration. Pure endoglucanase received sequential treatment with hydrophobic interactions and cation-exchange chromatography followed by gel-filtration. Endoglucanase activity of fractions collected during the purification was determined using as substrate carboxymethylcellulose (CMC) (procedure IUPAC 1987). Protein content was measured by a test Kit BioRad (Bio-Rad Laboratories), using bovine serum albumin as the standard.

The concentrated culture supernatant was applied to a column of hydrophobic vzaimodeystviya HiPrep 16/10 Butyl FF, balanced 20 mm potassium phosphate buffer pH of 6.0, containing 1 M (NH4)2SO4. Bound peroxidase proteins were suirable by a linear gradient from the above buffer and 5 mm potassium phosphate, pH to 6.0. Collected fractions and measured endoglucanase activity, as described above. Endoglucanase activity starred in a wide range of conductivity 120 - 15 SSM/see

Mixed fractions were applied to a cation-exchange column HiTrap SP XL, balanced 8 mm sodium acetate, pH 4.5. Bound peroxidase Proteus the s was suirable by a linear gradient from 0 to 0.25 M NaCl in equilibrium buffer. Protein having endoglucanase activity was suirable square conductivity 3-7 MS/see Cation-exchange chromatography was repeated, and protein eluate was concentrated by lyophilization.

The dissolved sample was loaded on the gel filtration column Superdex 75 HR 10/30, balanced 20 mm sodium phosphate buffer pH 7.0, containing 0.15 M NaCl. The main protein fraction was suirable with a column with a fixed volume of 13.3 ml Of the protein purity of the eluate was judged by electrophoresis on SDS-polyacrylamide gel, and estimated molecular weight of 40 kDa. The specific activity of the purified protein, designated as At EG_40, 50aboutWith determined as 450 ncat/mg (procedure IUPAC 1987, when used as a substrate CMC).

thermal stability of the purified endoglucanase was measured at different temperatures. The reaction was carried out in the presence of 0.1 mg/ml BSA at pH 5.0 for 60 min, using as substrate the carboxymethylcellulose. A. thermophilum EG-40/Cel5A was stable up to 80, up to 50 and up to 60aboutS, respectively. Control the enzymes from T. reesei EGI (Cel7B) and EGII (Cel5A) retained 100%activity 60 and 65aboutRespectively.

Example 4. Clean endoglucanase from Chaetomium thermophilum ALKO4261

Chaetomium thermophilum ALKO4261 were grown as described in Example 1. Pure endoglucanase received sequential treatment of hydrophobin the interactions and cation-exchange chromatography followed by gel-filtration. Endoglucanase activity of fractions collected during the purification was determined using as substrate carboxymethylcellulose (CMC) (procedure IUPAC 1987).

In the culture supernatant was added ammonium sulfate to achieve the same conductivity as in 20 mm potassium phosphate pH of 6.0, containing 1 M (NH4)2SO4. The sample was loaded on the column, hydrophobic interaction HiPrep 16/10 Phenyl FF, balanced 20 mm potassium phosphate buffer pH of 6.0, containing 1 M (NH4)2SO4. Elution was performed by a linear gradient from 20 to 0 mm potassium phosphate, pH of 6.0, with subsequent 5 mm potassium phosphate, pH 6.0 and water. Bound peroxidase proteins were suirable by a linear gradient from 0 to 8 M urea. Fractions were collected and analyzed endoglucanase activity, as described above. Protein having endoglucanase activity, elyuirovaniya at the beginning of the gradient of urea.

Mixed fractions were balanced 16 mm Tris-HCl pH 7.5 (I=1,4 MS/cm) using a 10DG columns (Bio-Rad) and used HiTrap DEAE FF anion exchange column, equilibrated to 20 mm Tris-HCl, pH 7.5. Bound peroxidase proteins were suirable by a linear gradient from 0 to 1 M NaCl in equilibrium buffer. Fractions were collected and analyzed for endoglucanase activity, as described above. The protein was suirable in the range of 10-20 MSM/see

The sample was balanced to 15 mm azeta the sodium, pH 4.5, using a 10DG columns (Bio-Rad) and used HiTrap SP XL cation-exchange column, equilibrated to 20 mm sodium acetate, pH 4.5. The protein was suirable by a linear gradient from 0 to 0.4 M sodium acetate, pH 4.5. Endoglucanase activity was filmed in the range of 1-10 MSM/see the Collected sample liofilizirovanny.

This sample is then dissolved in water and applied to a gel filtration column Superdex 75 HR 10/30, balanced 20 mm sodium phosphate, pH of 6.0, containing 0.15 M NaCl. Collected fractions and those that had endoglucanase activity was mixed. About the purity of the protein eluate was judged by electrophoresis on SDS-polyacrylamide gel and the molecular weight was estimated on the basis of molecular mass standards (standards for staining SDS-PAGE Broad Range, Bio-Rad) as a 54 kDa. pI of purified protein, designated as Ct EG_54, determined using the PhastSystem (Pharmacia) as approximately 5.5.

Example 5. Clean endoglucanase from Thermoascus aurantiacus ALKO4242

Thermoascus aurantiacus ALKO4242 were grown as described in Example 1. Pure endoglucanase received sequential purification by hydrophobic interaction and anion exchange chromatography followed by gel-filtration. Endoglucanase activity of fractions collected during the purification was determined using as substrate carboxymethylcellulose (CMC) (procedure IUPAC 1987). Protein content was measured by means of test Kit BioRad (Bio-Rad Laboratories), using as a standard bovine serum albumin.

The culture supernatant was applied to a column of hydrophobic vzaimodeystviya HiPrep 16/10 Butyl, balanced 20 mm potassium phosphate buffer pH 6.0 and containing 0.7 M (NH4)2SO4. Bound peroxidase proteins were suirable using 0.2 M (NH4)2SO4(I=39 MS/cm). Fractions having endoglucanase activity, mixed and concentrated by ultrafiltration.

The sample was absoluely on 10DG columns (Bio-Rad) and was applied to a HiTrap DEAE FF anion exchange column equilibrated with 15 mm Tris-HCl, pH 7.0. Bound peroxidase proteins were suirable by a linear gradient from 0 to 0.4 M NaCl in equilibrium buffer. Protein having endoglucanase activity was suirable square conductivity 15-21 MSM/see the Collected fractions were mixed and concentrated as described above.

The sample was applied on Sephacril S-100 HR 26/60 gel filtration column, equilibrated with 50 mm sodium acetate buffer, pH 5.0, containing 0.05 M NaCl. Protein fraction having endoglucanase activity was suirable with speakers with volume retention, corresponding to the molecular weight of 16 kDa. The collected fractions were mixed, concentrated and repeated gel filtration. About the purity of the protein eluate was judged by electrophoresis on SDS-polyacrylamide gel, and molecular weight was estimated as 28 kDa. pI of purified protein, the convoy is asenovo as Ta EG_28, determined by IEF-gel (PhastSystem, Pharmacia) as 4290 ncat/mg (procedure IUPAC 1987, using CMC as substrate).

Example 6. Purification and characterization of β-glucosidase from Acremonium thermophilum ALKO4245

Acremonium thermophilum ALKO4245 were grown as described in Example 1. Pure β-glucosidase received sequential purification by hydrophobic interaction and anion exchange chromatography followed by gel-filtration. β-glucosidase activity of fractions collected during the purification was determined using 4-nitrophenyl-β-D-glucopyranoside as substrate (Bailey and Linko, 1990). Protein content was measured using the test Kit BioRad (Bio-Rad Laboratories), using as a standard bovine serum albumin.

The culture supernatant was applied to a HiPrep 16/10 Phenyl Sepharose FF column, hydrophobic interaction, balanced 20 mm potassium phosphate, pH of 6.0, containing 1 M (NH4)2SO4. Bound peroxidase proteins were suirable by a linear gradient from the equilibrium buffer and 5 mm potassium phosphate field conductivity 137-16 MSM/see the Collected fractions were mixed and concentrated by ultrafiltration.

The sample was absoluely in 10DG-columns (Bio-Rad) and was applied to a HiTrap DEAE FF anion exchange column equilibrated with 10 mm potassium phosphate, pH 7.0. Bound peroxidase protein a elution by a linear gradient from the equilibrium buffer before the e of the buffer, containing 0.25 M NaCl in the field conductivity of 1.5-12 MSM/see Anion-exchange chromatography was repeated, as described above, except that used 4 mm potassium-phosphate buffer, pH 7,2. Proteins were suirable field conductivity 6-9 MSM/see Faction with β-glucosidase activity were collected, mixed and concentrated.

Active material with anion-exchange chromatography was applied to a column Sephacril S-300 HR 26/60, balanced 20 mm potassium phosphate, pH 6.5, containing 0.15 M NaCl. Protein with β-glucosidase activity was suirable with sufficient retention volume corresponding to a molecular weight 243 kDa. The protein was considered to be pure according to the results of electrophoresis on SDS-polyacrylamide gel. pI of purified protein, designated At βG_101, was determined by IEF-gel (PhastSystem, Pharmacia) in the field of 5.6 and 4.9. Specific activity At βG_101 at 50aboutWith determined 1100 ncat/mg (using 4-nitrophenyl-β-D-glucopyranoside as substrate) (Bailey and Linko, 1990).

thermal stability of the purified β-glucosidase was determined at different temperatures. The reaction was carried out in the presence of 0.1 mg/ml BSA at pH 5.0 for 60 min, 4-nitrophenyl-β-D-glucopyranoside as substrate. A. thermophilum βG_101 remained stable up to 70aboutControlmy enzyme Aspergilus (Novozym 188) maintained a 100-percent activity up to 60aboutC.

Example 7. Purification of β-glucosidase from Chaetomium thermophilum ALKO4261

Chaetomium thermophilum ALKO461 grew, as described in Example 1. Pure β-glucosidase received sequential purification by hydrophobic interaction, anion - and cation-exchange chromatography followed by gel-filtration. β-glucosidase activity of fractions collected during the purification was determined using 4-nitrophenyl-β-D-glucopyranoside as substrate (Bailey and Linko, 1990).

The culture supernatant was applied to a HiPrep 16/10 Phenyl Sepharose FF column, hydrophobic interaction, balanced 20 mm potassium phosphate, pH 6.0 and containing 0.8 M (NH4)2SO4. Elution was performed by a linear gradient from the equilibrium buffer and 3 mm potassium phosphate, pH to 6.0, followed by elution with water and 6 M urea. The first fraction with β-glucosidase activity was suirable field conductivity 80-30 MSM/see Second fraction with β-glucosidase activity, elyuirovaniya urea, collected and absoluely on 10DG-columns (Bio-Rad), equilibrated with 10 mm Tris-HCl, pH 7.0.

After desalting the sample was applied to a HiTrap DEAE FF anion exchange column equilibrated with 15 mm Tris-HCl, pH 7.0. Protein not contacted with the column and elyuirovaniya at the time of filing of the sample. This flow-through fraction was absoluely on 10DG-columns (Bio-Rad), equilibrated 7 mm Na acetate, pH 4.5.

The sample with anion-exchange chromatography was applied to a HiTrap DEAE FF cation-exchange column, equilibrated with 10 mm sodium acetate, p 4,5. Bound peroxidase protein a elution by a linear gradient from 10 to 400 mm sodium acetate, pH 4.5. Fraction with β-glucosidase activity, erwerbende field conductivity of 6.5-12 MS/cm, harvested, absoluely on 10DG-columns (Bio-Rad), equilibrated 7 mm sodium acetate, pH 4.5, and liofilizirovanny.

The lyophilized sample was diluted to 100 ál with water and was applied to a gel filtration column Superdex 75 HF 10/30, balanced 20 mm sodium phosphate, pH 4.5, containing 0,15 NaCl. β-glucosidase activity was lirowaus when the confinement volume 13,64 ml of the Collected fractions was mixed, liofilizirovanny and dissolved in water. About the purity of the protein was assessed by electrophoresis on SDS-polyacrylamide gel, and molecular weight was estimated as 76 kDa. The protein identified as Ct βG_76.

Example 8. Purification and characterization of β-glucosidase from Thermoascus aurantiacus ALKO4242

Thermoascus aurantiacus ALKO4242 were grown as described in Example 1. Pure β-glucosidase received sequential purification by hydrophobic interaction, anion - and cation-exchange chromatography followed by gel-filtration. β-glucosidase activity of fractions collected during the purification was determined using 4-nitrophenyl-β-D-glucopyranoside as substrate (Bailey and Linko, 1990). Protein content was measured using the test Kit BioRad (Bio-Rad Laboratories), using as a standard the bullish Siva otechny albumin.

The culture supernatant was applied to a column of hydrophobic vzaimodeystviya HiPrep 16/10 Fenyl Sepharose FF equilibrated to 20 mm potassium phosphate, pH 6.0 and containing 0.7 M (NH4)2SO4. Bound peroxidase proteins were suirable by a linear gradient from 0.2 M (NH4)2SO4up to 5 mm potassium phosphate, pH to 6.0. β-glucosidase activity was suirable within the gradient field conductivity 28,0-1,1 MSM/see Fractions were mixed and concentrated by ultrafiltration.

The sample was absoluely on 10DG columns (Bio-Rad) and was applied to a HiTrap DEAE FF anion exchange column, equilibrated to 20 mm Tris-HCl, pH 7.0. The protein was suirable by a linear gradient from 0 to 0.2 M NaCl in equilibrium buffer and detained by elution with 20 mm Tris-HCl containing 0.4 M NaCl. Sample suirvey field conductivity of about 10-30 MS/cm, was concentrated by ultrafiltration and absoluely using a 10DG columns (Bio-Rad).

The sample was applied to a HiTrap SP XL cation-exchange column, equilibrated 9 mm sodium acetate, pH 4.5. The enzyme was suirable by a linear gradient from 10 to 400 mm NaAc and using the delayed elution using 400 mm NaAc, pH 4.5. Proteins with β-glucosidase activity was suirable for a linear gradient in the conductivity of 5.0 to 11.3 MSM/see

Active material with a cation-exchange chromatography was applied to a column Sephacril S-300 HR 26/60, uravnovesen the Yu 20 mm sodium phosphate, pH 7.0, containing 0.15 M NaCl. Protein with β-glucosidase activity was suirable with sufficient retention volume corresponding to a molecular weight of 294 kDa. The collected fractions were mixed, liofilizirovanny and dissolved in water. About the purity of the protein was assessed by electrophoresis on SDS-polyacrylamide gel, and molecular weight was estimated as 81 kDa, representing very likely manometrically form of the protein. Isoelectric focusing (IEF) was performed using 3-9 pI-gel. After staining with silver in addition to a narrow band corresponding to pI 4,55, stained extensive area above the pI of 5.85. The specific activity of the dyed protein, designated as Ta βG_81, 50aboutWith determined as 600 ncat/mg, using 4-nitrophenyl-β-D-glucopyranoside as substrate (Bailey and Linko, 1990).

thermal stability of the purified β-glucosidase was determined at different temperatures. The reaction was carried out in the presence of 0.1 mg/ml BSA at pH 5.0 for 60 min, using as substrate 4-nitrophenyl-β-D-glucopyranoside. T. aurantiacus βG_81 was stable up to 75aboutC. Controlling enzyme Aspergilus (Novozym 188) retained 100%activity 60aboutC.

Example 9. Purification of xylanase from Acremonium thermophilum ALKO4245

Acremonium thermophilum ALKO4245 were grown as described in Example 1. The supernatant of the culture was incubated at 70aboutC for 24 hours, after which it was concentrated in what travelitaly. Pure xylanase received sequential purification by hydrophobic interactions and cation-exchange chromatography followed by gel-filtration. Xylanase activity was determined using birch xylan as substrate (procedure IUPAC 1987). The protein was analyzed using the test Kit BioRad (BioRad Laboratories), using as a control, bovine serum albumin.

The concentrated culture supernatant was applied to a hydrophobic interaction column HiPrep 16/10 Butyl FF, balanced 20 mm potassium phosphate buffer, pH of 6.0, containing 1 M (NH4)2SO4. Bound peroxidase proteins were suirable by a linear gradient from the above buffer and 5 mm potassium phosphate, pH to 6.0. Protein fraction was suirable in the wide field conductivity of 5.0 to 11.3 MSM/see

Sample with a hydrophobic interaction column was applied to a HiTrap SP XL cation-exchange column, equilibrated 8 mm sodium acetate, pH 4.5. The protein was not associated with this column and elyuirovaniya in the stream during loading of the sample. The eluate was concentrated by ultrafiltration. Hydrophobic chromatography was repeated as described above. Unbound proteins were collected and liofilizirovanny.

The dissolved sample was loaded on the gel filtration column Superdex 75 HR 10/30, balanced 20 mm sodium phosphate buffer, pH 7.0, containing 0.15 to NaCl. P is otein, suirvey with a column with sufficient retention volume of 11.2 ml, was judged to be pure by gel electrophoresis on SDS-polyacrylamide gel. The molecular mass of the purified protein was estimated on the basis of molecular mass standards (standards for staining SDS-PAGE Broad Range, Bio-Rad) as a 60 kDa. The specific activity of the protein, designated as At XYN_60, 50aboutWith defined in 1800 ncat/mg (procedure IUPAC 1987, using birch xylan as substrate). The relative activity was increased from 1.2 times at 60aboutWith up to 1.6 times in the 70aboutCompared to the 50aboutC. Specific activity against MUG2 (4-methylumbelliferyl-β-D-cellobioside), MUL (4-methylumbelliferyl-beta-D-lactose) and MUG3 (4-methylumbelliferyl-β-D-relationid) 54, 33, and 78 ncat/mg (50aboutC, pH 5.0, for 10 min), respectively. This is consistent with the fact that the family 10 xylanases also shows activity against arylpropanoids (Biely et al., 1997).

Example 10. Purification of xylanase from Thermoascus aurantiacus ALKO4242

Thermoascus aurantiacus ALKO4242 were grown as described in Example 1. Pure xylanase received sequential purification by hydrophobic interaction, anion - and cation-exchange chromatography followed by gel-filtration. Xylanase activity was determined using birch xylan as substrate (procedure IUPAC 1987). Protein Ana who was seravalli using the test Kit BioRad (Bio-Rad Laboratories), using as a control, bovine serum albumin.

The culture supernatant was applied to a hydrophobic interaction column HiPrep 16/10 Phenyl Sepharose FF equilibrated to 20 mm potassium phosphate buffer, pH 6.0 and containing 0.7 M (NH4)2SO4. Bound peroxidase proteins were suirable through a two-stage procedure. Elution was performed by lowering the salt concentration at first to 0.2 M (NH4)2SO4and then by a linear gradient from 20 mm potassium phosphate, pH of 6.0, containing 0.2 M (NH4)2SO4up to 5 mm potassium phosphate, pH to 6.0. The protein was suirable using 0.2 M (NH4)2SO4(I=39 MS/cm).

The sample was absoluely on 10DG columns (Bio-Rad) and was applied to a HiTrap DEAE FF anion exchange column equilibrated with 15 mm Tris-HCl, pH 7.0. Protein not contacted with the anion exchange column and was elyuirovaniya in the stream. The conductivity of the sample was regulated in accordance with a conductivity of 20 mm sodium acetate, pH 4.5, adding water, and the pH was adjusted to 4.5 during concentration by ultrafiltration.

The sample was applied to a HiTrap SP XL cation-exchange column, equilibrated to 20 mm sodium acetate, pH 4.5. Bound peroxidase proteins were suirable by a linear gradient from the equilibrium buffer to the same buffer containing 1 M NaCl. The enzyme was suirable field conductivity 1-7 MSM/see Sample liofilizirovanny and p is after this was dissolved in water.

The lyophilized sample was dissolved in water and applied to a gel filtration column Superdex 75 HR 10/30, balanced 20 mm sodium phosphate, pH 7.0, containing 0.15 to NaCl. The protein was suirable with a column with sufficient retention volume corresponding to a molecular weight of 26 kDa. The protein was judged to be pure by gel electrophoresis on SDS-polyacrylamide gel. The molecular weight of the pure protein was estimated on the basis of molecular mass standards (standards for staining SDS-PAGE Broad Range, Bio-Rad). pI of purified protein, designated as Ta XYN_30, determined using the PhastSystem (Pharmacia)as approximately equal to 6.8. The specific activity of the Ta XYN_30 at 50aboutWith determined equal to 4800 ncat/mg (procedure IUPAC 1987, using birch xylan as substrate).

Example 11. Internal amino acid sequencing

Internal peptides sequenced using electrospray ionization combined with tandem mass spectrometry (ESI-MS/MS), using the device Q-TOF1 (Micromass). Protein first alkilirovanie and was divided into trypticase peptides. The resulting peptides were absoluely and partially separated using liquid nanochromatography (reversed phase) before use of the device Q-TOF1. Internal sequences of the peptides Chaetomium thermophilum and Acremonium thermophilum shown in Table 2. Peptides from Chaetomium CBH were received after the relevant cbh gene was cloned. Peptides derived from Acremonium CBH, was not used in the cloning of the corresponding gene.

Table 2
Internal sequences of peptides derived from Chaetomium thermophilum ALKO4265 CBH (1_C-4) and Acremonium thermophilum ALKO4245 CBH (1_A-4_A)
PeptideSequence
Peptide ST P S T N D A N A G F G R
Peptide SV A F S N T D D F N R
Peptide SF S N T D D F N R K
Peptide SP G N S L I T E Q Y C D A Q/K K
Peptide AV T Q F I/L T G
Peptide AM G D T S F Y G P G
Peptide AC D P D G C D F N
Peptide AS G N S L I T T D F
I/L = leucine and isoleucine have the same molecular mass and cannot be distinguished by ESI-MS/MS analysis;
Q/K = molecular weight of glutamine and lysine differs only in being 0.036 Yes, so they can't do is be distinguished by ESI-MS/MS analysis.

The internal consistency of purified endoglucanases, β-glucosidase and xylanases Acremonium thermophilum ALKO4245, Chaetomium thermophilum ALKO4261 and Thermoascus aurantiacus ALKO4242 are shown in Tables 3-5.

Table 3
Internal sequences of peptides derived from endoglucanases Acremonium thermophilum ALKO4245 EG_40, Chaetomium thermophilum ALKO4261 EG_54 and Thermoascus aurantiacus ALKO4242 EG_28
ProteinPeptideSequence(a
At EG-40Peptide 1Q S C S S F P A P L K P G C Q W R
Peptide 2Y A L T F N S G P V A G K
Peptide 3V Q C P S E L T S R
Peptide 4N Q P V F S C S A D W Q R
Peptide 5Y W D C C K P S C G W P G K
Peptide 6P T F T
Ct EG_54Peptide 1 E P E P E V T Y Y V
Peptide 2Y Y L L D Q T E Q Y
Peptide 3R Y C A M D L W E A N S R
Peptide 4P G N T P E V H P Q / K
Peptide 5S I /L A P P H C N Q / K
Peptide 6Q Q Y E M F R
Peptide 7A L N D D F C R
Peptide 8W G N P P P R
Ta EG_28Peptide 1I /L T S A T Q W L R
Peptide 2G I C A /L S A T C V S S T I /L G Q E R
Peptide 3P F M M E R
Peptide 4Q Y A V V D P H N Y G R
(a I/L = leucine and isoleucine have the same molecular mass and cannot be distinguished by means of the PTO ESI-MS/MS analysis Q/K = molecular weight of glutamine and lysine differs only in being 0.036 Yes, so they cannot be distinguished by ESI-MS/MS analysis.

Table 4
Internal sequences of peptides derived from beta-glucosidase Acremonium thermophilum ALKO4245 βG_101, Chaetomium thermophilum ALKO4261 βG_76 and Thermoascus aurantiacus ALKO4242 βG_81
ProteinPeptideSequence(a
At βG_101Peptide 1S P F T W G P T R
Peptide 2V V V G D A D G N P C
Peptide 3A F V S Q L T L L E K
Peptide 4G T D V L /I Y T P N N K
Peptide 5Q P N P P A G N A C V L /I R
Ct βG_76Peptide 1E G L F I D Y R
Peptide 2P G Q S G T A T F R
Peptide 3E T M S N S V D D R
Peptide 4I A L V a G S A A V V
Peptide 5M W L C E N D R
Peptide 6Y P Q L C L Q D G P L G I R
Peptide 7E L N G Q N S G Y P S I
Ta βG_81Peptide 1T P F T W G K
Peptide 2L C L Q D S L P G V R
Peptide 3G V D V Q L G P V A G V A P R
Peptide 4V N L T L E
Peptide 5F T G V F G E D V V G
Peptide 6N D L P L T G Y E K
(a I/L = leucine and isoleucine have the same molecular mass and cannot be distinguished by ESI-MS/MS analysis.

Table 5
Internal sequences of peptides derived from beta-glucosidase Acremonium thermophilum ALKO4245 XYN_60 and Thermoascus aurantiacus ALKO4242 XYN_30
ProteinPeptideSequence
At XYN_60Peptide 1Y N D Y N L E Y N Q K
Peptide 2F G Q V T P E N
Peptide 3V D G D A T Y M S Y V N N K
Peptide 4K P A W T S V S S V L A A K
Peptide 5S Q G D P R I V A K
Ta XYN_30Peptide 1V I F G V A D Q N R
Peptide 2N A A I I Q A D F G Q V T P E N S M a K
Peptide 3G H T L V W H S Q L P S W V S S I T D K
Peptide 4N H I T T L M T R
Peptide 5A W D V V N E A F N E D G S L R
Peptide 6L Y I N D Y N L D S A S Y P K
Peptide 7A S T T P L F D G N F N P K P A Y N A I V Q D L Q Q
Peptide 8Q T V F L N a V I G E D Y I P I A F Q T A R

Example 12. Construction of genomic libraries for Thermoascus aurantiacus, Chaetomium thermophilum and Acremonium thermophilum

Genomic library Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245 created in the vector Lambda DAS®II (Stratagene, USA) according to instructions from the supplier. Chromosomal DNA extracted by the method of RV and Broda (1985), was partially digested with Sau3A. Cleaved DNA was fractionally in size and fragments of a selected size (≈5-23 KBP) ligated with BamHI-shoulders split lambda vector. Legirovannye mixture was packaged using packaging extracts Gigapack III Gold in accordance with the manufacturer's instructions (Stratagene, USA). Titles genomic libraries Chaetomium thermophilum and Acremonium thermophilum was 3.6×106PFU (plaque-forming units)/ml and 3.7×105PFU/ml, and the titles of their amplified libraries to 6.5×1010PFU/ml and 4.2×108PFU/ml, respectively.

When konstruirovanie the genomic libraries Thermoascus aurantiacus ALKO4242 and Chaetomium thermophilum ALKO4261 used Set of Lambda FIX® II/Xpo I Partial Fill-In Vector (Stratagene, USA) in accordance with instructions from the supplier. Chromosomal DNA extracted by the method of RV and Broda (1985), was partially digested with Sau3A. Cleaved DNA was fractionally in size and fragments of a selected size (≈ 6-23 KBP) was inserted and ligated with XhoI-shoulders split Lambda FIX II vector. Legirovannye mixture was packaged using packaging extracts Gigapack III Gold in accordance with the manufacturer's instructions (Stratagene, USA). Titles genomic libraries Thermoascus aurantiacus ALKO4242 and Chaetomium thermophilum ALKO4261 was 0.2×106PFU/ml and 0.3×106PFU/ml, and the titles of their amplified libraries of 1.8×109PFU/ml and 3.8×109PFU/ml, respectively.

Example 13. Cloning genes cellobiohydrolase (cbh/cel7) from Thermoascus aurantiacus, Chaetomium thermophilum and Acremonium thermophilum

When the selection and processing of DNA (plasmids, DNA fragments) enzymes, when tranformirovanie E.coli, etc. used standard methods of molecular biology. The main methods used are described in the standard manuals of molecular biology, for example, Sambrook et al. (1989) and Sambrook and Russel (2001).

Probes for screening genomic libraries were constructed as described in Example 12, amplified by PCR, using reactions as matrices genomic DNA Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245. In accordance with the published nucleotide n is a sequence of (WO 03/000941, Hong et al., 2003b) was designed several primers tested in PCR reactions. PCR reaction mixture contained 50 mm Tris-HCl, pH of 9.0, 15 mm (NH4)2SO4, with 0.1 Triton X-100, 1.5 mm MgCl2, 0.2 mm dNTPs, 5 μm of each primer and 1 unit of Dynazyme EXT DNA polymerase (Finnzymes, Finland), and ≈0.5-1 ág of genomic DNA. Conditions for PCR reactions were as follows: 5 min initial denaturation at 95aboutC, then 30 cycles of 1 min at 95aboutWith either 1 min of annealing at 62about(Difference ±8about(C) for matrices Thermoascus ALKO4242 and Chaetomium ALKO4265, or 1 min annealing at 58about(Difference ±6about(C) for the matrix Acremonium ALKO4245, elongation for 2 min at 72aboutC and final elongation at 72aboutC for 10 minutes

The DNA products of the expected sizes (calculated from published cbh-sequences) were obtained from all of the used matrices. DNA fragments of the expected size were isolated from the most specific PCR reaction and cloned in the vector pCR®Blunt-TOPO®(Invitrogen, USA). Inserts were characterized by sequencing and holding Southern blot-hybridisable with genomic DNA cleaved several restrictionenzyme enzymes. The PCR fragments that were selected for use as probes for screening genomic libraries Thermoascus aurantiacus, Chaetomium thermophilum and Acremonium thermophilum, presented in Table 6.

Table 6
The primers used in PCR reactions, and the probes selected for screening cbh/cel7 genes from genomic libraries Thermoascus aurantiacus, Chaetomium thermophilum and Acremonium thermophilum. Shows genomic DNA templates and the names of the plasmids containing probe fragments.
GeneDirect primerReverse primerMatrix DNAThe DNA fragment (KBP)Plasmid
Ta cbhTCEL11
atgcgaactggcgttgggtcc
TCEL12
gaatttggagctagtgtcgacg
Thermoascus ALKO42420,8pALK1633
Ct cbhTCEL7
cgatgccaactggcgctggac
TCEL8
ttcttggtggtgtcgacggtc
Chaetomium ALKO42650,8pALK1632
At cbhTCEL13
agctcgaccaactgctacacg
TCEL4
accgtgaacttcttgctggtg
Acremonium ALKO42450,7pALK1634

Derived from all of these probes amino acids of the s sequences had homology with several published CBH sequences (BLAST program, version 2.2.9 in NCBI, the National Center for Biotechnology Information; Altschul et al., 1990) glycosylglycerols family 7 (Henrissat, 1991; Henrissat and Bairoch, 1993).

Inserts of the plasmids listed in Table 6, were marked by digoxigenin in accordance with the supplier's instructions (Roche, Germany), and amplificatoare genomic library (plaque 2×105-3×105) was skanirovali labeled probe fragments. The temperature of hybridization on filters was 68aboutWith, and the filters were washed 2×5 min at RT using 2×SSC and 0.1% SDS, followed 2×15 min at 68aboutWith using of 0.1×SSC and 0.1% SDS using homologous probes. From each hybridization were received several positive plaques. When skanirovaniya genomic libraries Acremonium ALKO4245 some of the positive plaques were strongly hybridisable with isprobuem probe, but, in addition, there were a number of plaques, which was weaker hybridizations with probes. It was assumed that in the genome may have been present(s) other(s) cellobiohydrolase(s) gene(s), caller(s) can cross-react. When skanirovaniya genomic libraries Thermoascus ALKO4242 and Chaetomium ALKO4265 was purified from four to five strongly gibridizatsiya plaques. In the case of Acremonium ALKO4245 four of the five purified plaques were weakly hybridized used by the probe. Phage DNA was isolated and characterized using Southern blot-hybridisable. Wybran the e restriction fragments, hybridities probe was subcloned into the vector pBluescript II KS+, and relevant areas of the clones were sequenced.

The result was cloned four cbh/cel7 gene; one from Thermoascus aurantiacus ALKO4242, one of the Chaetomium thermophilum ALKO4265 and two of Acremonium thermophilum ALKO4245 (early works they had symbols At_cbh_C and At_cbh_A, and then paleobotany as At cel/7A and At cel/7B, respectively). Table 7 summarizes information about the probes used for screening genes, phage clones, which were selected genes selected restriction fragments containing the genes for the complete length with their promoter and termination regions, the names of the plasmids and Deposit numbers DSM for E. coli strains carrying these plasmids.

Table 7
The probes used for cloning cbh/cel7 genes, phage clone selected subclones, the number plasmids and number of the Deposit of the appropriate strain of E.coli
GeneThe probe used for screeningPhage cloneFragment, subcloned in pBluescript IIPlasmid No.E.coli Depository No.
Ta cel7A pALK1633F12the 3.2 KBP XbaIpALK1635DSM 16723
Ct cel7ApALK1632F36the 2.3 KBP PvuI-HindIIIpALK1642DSM 16727
At cel7BpALK1634F6a 3.1 KBP EcoRIpALK1646DSM 16728
At cel7ApALK1634F2the 3.4 KBP XhoIpALK1861DSM 16729

Relevant information on genes and deduced protein sequences (SEQ ID nos: 1-8) are summarized in Table 8 and 9, respectively.

The peptide sequence of the purified CBH protein from Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245 (table 2) were found from the deduced amino acid sequences of clones containing Ct cel7A and At cel7A-genes. Thus, we can conclude that the genes encoding the purified CBH/Cel7 proteins from Chaetomium thermophilum and Acremonium thermophilum, were cloned.

Table is CA 8
Summary of cbh/cel7-genes isolated from Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245
Cbh geneThe length of the introns (BP)(aThe coding region (BP)(bThe number of intronsThe length of introns (BP)SEQ ID NO:
Ta cel7A143913711651
Ct cel7A166315961647
At cel7B172213773134, 122, 873
At cel7A18531569488, 53, 54, 865
(a STOP codon is included.
(b - a STOP codon is not included.

Table 9
Summary of amino acid sequences derived from the sequences of the cbh/cel7 genes from Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245. ss, signal sequence
CBH proteinNumber of aminos.Length ss NN/HMM(aC-end-howl CBD(bThe predicted MW (Da, ss excl.)(cThe predicted pI (ss excl.)The alleged sites of N-glycosylation(dSEQ ID NO:
Ta Cel7A45717/17NO46873of 4.4422
Ct Cel7A53218/18YES, T497-L53254564of 5.0538
At Cel7B45921/21NO/td> 47073a 4.8324
At Cel7A52317/17YES, Q488-L523536964,6746
(a Prediction of the signal sequence was made using the program SignalP V3.0 (Nielsen et al., 1997; Bendtsen et al., 2004); the values of NN were obtained using the neural circuit, and the values HMM, using hidden modules Markov.
(b - Pulp-binding domain (CBD), indicated amino acids C-terminal region CBD (M1 (Met #1) included in the number/
(c - Predicted signal sequence is not included. The prediction was made using the Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003).
(d is the Number of sequences N-X-S/T.

The deduced amino acid sequence of Thermoascus aurantiacus Cel7A and Acremonium thermophilum Cel7A (short, without CBD) were highly homologous to one another (analyzed General alignment Needleman-Wunsch, EMBOSS 3.0.0 Needle c EBLOSUM62 matrix, the penalty for omission of 10.0 and an extension penalty of 0.5; Needleman ans Wunsch, 1970). In addition, the deduced sequence Cel7A Acremonium thermophilum had lower identity with the derived p is a sequence of Cel7A Chaetomium thermophilum. Acremonium thermophilum Cel7 In rather different from CBH/Cel7 sequences according to the invention.

Deduced sequence Cel7A Chaetomium had the highest identity (analyzed General alignment Needleman-Wunsch, EMBOSS Needle, see above) with the polypeptides CBHI Chaetomium thermophilum, Scytalidium thermophilum and Thielavia australiensis, described in WO 03/000941. Similarly deduced sequence Cel7A Thermoascus aurantiacus was highly identical to the published CBHI Thermoascus aurantiacus (WO 03/000941, Hong et al., 2003b). Cel7 In Acremonium thermophilum had significantly lower identity to previously published sequences, being more closely associated with CBHI-polypeptide from Oryza sativa. The highest homology in the derived sequence Cel7A Acremonium thermophilum were Exidia gladulosa and CBHI-polynucleotide Acremonium thermophilum (WO 03/000941). The alignment indicates that the cloned sequence ALKO4242 Thermoascus aurantiacus, ALKO4265 Chaetomium thermophilum and ALKO4245 Acremonium thermophilum encode CBH-proteins having high homology with the polypeptides of glycosidically family of 7, which for this reason has received the designation Cel7A or Cel7 (Henrissat et al., 1998).

Comparison of the deduced sequences cbh/cel7 genes from Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245 with one another and next to the sequences found in the databases shown in Table 10.

Table 10
The most homologous sequence with the deduced amino acid sequences of the cbh/cel7 genes from Thermoascus aurantiacus ALKO4242, Chaetomium thermophilum ALKO4265 and Acremonium thermophilum ALKO4245. The alignment was performed using the total alignment Needleman-Wunsch, (EBLOSUM62, the penalty for omission of 10.0 and an extension penalty of 0.5). *indicates the amino acid sequence originating from one of cellobiohydrolase genes cloned in this work. "Cor" specifies the alignment without CBD
The body, the enzyme and the access numberIdentity %
* Thermoascus aurantiacus Cel7A100,0
Thermoascus aurantiacus, AY84098299,6
Thermoascus aurantiacus, AX657575of 99.1
Thermoascus aurantiacus, AF421954of 97.8
Talaromyces emersonii, AY08176679,5
Chaetomidium pingtungium, AX65762376,4
Ttichophaea saccata, AX65760773,4
* Acremonium thermophilum Cel7A (KOR)70,6
Emericella nidulans, AF420020 (KOR) 70,4
* Chaetomium thermophilum Cel7A (KOR)66,4
* Chaetomium thermophilum Cel7A100,0
Chaetomium thermophilum, AY86134791,9
Chaetomium thermophilum, AX65757191,7
Scytalidium thermophilum, AX657562774,7
Thielavia australiensis, AX65757774,6
Acremonium thermophilum, AX65756972,3
Exidia glandulosa, AX65761368,0
* Acremonium thermophilum Cel7A66,9
* Thermoascus aurantiacus Cel7A (KOR)66,4
Exidia glandulosa, AX65761560,8
Chaetomium pingtungium, AX65762360,7
* Acremonium thermophilum Cel7B (KOR)60,2
* Acremonium thermophilum Cel7B100,0
Oryza sativa, AK10894866,1
Exidia glandulosa, AX65761565,0
Acremonium thermophilum, AX657569 (KOR) 64,8
Thermoascus aurantiacus, AX65757564,8
* Acremonium thermophilum Cel7A64,6
* Thermoascus aurantiacus Cel7A64,4
Ttichophaea saccata, AX65760763,6
* Chaetomium thermophilum Cel7A (KOR)60,2
* Acremonium thermophilum Cel7A100,0
Exidia glandulosa, AX65761377,9
Exidia glandulosa, AX65761577,9
Acremonium thermophilum, AX65756977,5
Thielavia australiensis, AX65757771,0
* Thermoascus aurantiacus Cel7A (KOR)70,6
Scytalidium thermophilum, AX65762767,5
Chaetomium thermophilum, AX65757167,5
Chaetomium pingtungium, AX65762367,3
* Chaetomium thermophilum Cel7A66,9
* Acremonium thermophilum Cel7B (KOR)64,6

Example 14. PR is dotirovanie recombinant CBH/Cel7-proteins in Trichoderma reesei

Expression plasmids designed to obtain recombinant CBH/Cel7-proteins from Thermoascus aurantiacus (Ta Cel7A), Chaetomium thermophilum (Ct Cel7A) and Acremonium thermophilum (At Cel7A, At Cel7B; early works of these proteins had a time designation At CBH_C and At CBH_A, respectively). Constructed expression plasmids are listed in Table 11. Recombinant cbh/cel7 genes, including their own signal sequences, were exactly fused to the T. reesei cbh1 (cel7A)promoter by PCR. Termination of transcription was provided by T. reesei cel7A-terminated, and for selection of transformants was used A. nidulans amdS marker gene, as described by Paloheimo et al. (2003). The linearized expression cassette (Figure 2) was isolated from vector frames after splitting EcoRI and transformed into T. reesei A96 motorway and a-protoplasts (both strains were deleterows the genes encoding the four major cellulase CBHI/Cel7A, CBHII/Cel6A, EGI/Cel7B and EGII/Cel5A). Transformation was carried out as Penttilä et al. (1987) with modifications described by the Karhunen et al. (1993), making selection with ndimethylacetamide as the sole nitrogen source. Transformants were purified on selective cups through a single conidia to their sporulation on PD.

/tr>
Table 11
Expression cassettes, construire is installed for producing CBH/Cel7-protein Thermoascus aurantiacus ALKO4242 (Ta Cel7A), Chaetomium thermophilum ALKO4265 (Ct Cel7A) and Acremonium thermophilum ALKO4245 (At Cel7A, At Cel7B) in Trichoderma reesei. The General structure of the expression cassettes was as shown in figure 2. Cloned cbh/cel7 genes were exactly fused to the T. reesei cbh1/cel7A-promoter
CBH/Cel7Expression plasmidSize expr. cartridge(andcel7A terminator(b
Ta Cel7ApALK18519,0 KBP245 BP (XbaI)
Ct Cel7ApALK1857of 9.2 KBP240 BP (HindIII)
At Cel7BpALK1860of 9.4 KBP361 BP (EcoRI)
At Cel7ApALK1865the 9.5 KBP427 BP (EcoRV)
(a - Expression cassette for transformation of T. reesei was isolated from the vector frame using cleavage with EcoRI.
(b is the Number of nucleotides of the genomic termination region cbh1/cel7A after the STOP codon. Restriction site on the 3'-end, used during the excision of the gene fragment of the genome, enclosed in parentheses.

CBH/Cel7 the production of the transformants was analyzed by supernatants crops cultivated in shake flasks (50 ml). Transformants were grown for 7 days at 28aboutWith in the complex based on the lactose inducing cellulase environment (Joutsjoki et al. (1993), buferiruemoi 5% KH2PO4. Cellobiohydrolase activity was analyzed using 4-methylumbelliferyl-β-D-lactoside (MUL) substrate in accordance with van Tilbeurgh et al., 1988. The genotypes of the selected transformants was confirmed using Southern-blots, in which was included a few genomic hydrolization, and appropriate expression cassette was used as a probe. Heterologous expression of Ta Cel7A-, Ct Cel7A-At Cel7A and At Cel7B protein was analyzed using SDS-PAGE and subsequent staining of Kumasi. The discovery that no cellobiohydrolase activity or production of a heterologous protein in SDS-PAGE is not found, suggests that when using the experimental method described At Cel7B is produced in Trichoderma below levels of detection.

The recombinant CBH/Cel7 enzymes are characterized in terms of pH optimum and thermal stability. the pH optimum of the recombinant CBH/Cel7-proteins from Thermoascus aurantiacus, Chaetomium thermophilum and Acremonium thermophilum was determined in a universal buffer of MacLaine in the range of pH 3.0 to 8.0, using the 4-methylumbelliferyl-β-D-lactoside (MUL) as substrate (Figa). the pH optimum for Ct Cel7A and At Cel7A is 5.5, above which the activity is starting to fall. the pH optimum of the recombinant crude Ta Cel7A - 5,0 (Figa). Thermal stability of recombinant Cel7 enzymes was determined by measuring MUL-activity in universal buffer of MacLaine at the optimum pH with reaction time of 1 h As shown by the results, Ta Cel7A and Ct Cel7A maintained for more than 60% of its activity at 70aboutWith, while At Cel7A showed clearly less stability at higher temperatures (≥65about(C) (Pigv).

Selected CBH/Cel7-transformants were cultured in laboratory bioreactors in the 28aboutWith the above environment within 3-4 days with pH control, 4,4±0,2 (NH3/H3PO4to get material for tests on the application. Supernatant recovered by centrifugation and filtration through a Seitz-150 K and EK-filters (Pall SeitzSchenk Filtersystem GmbH, Bad Kreuznach, Germany).

Example 15. Production of recombinant Thermoascus aurantiacus Cel7A+CBD fusion proteins in T. reesei

Thermoascus aurantiacus Cel7A (AF478686, Hong et al., 2003b; SEQ ID NO: 1) was merged with the linker and CBD Trichoderma reesei CBHI/Cel7A (AR088330, Srisodsuk et al., 1993) (= Tr CBD) with the subsequent receipt of the fusion protein (SEQ ID NO:28, the corresponding nucleic acid is SEQ ID NO:27) in T. reesei, as described in FI20055205/US 11/119,526; filed April 29, 2005. In addition, Thermoascus aurantiacus Cel7A was merged with the linker and CBD Chaetomium thermophilum Cel7A (SEQ ID NO:7) (Ct CBD). To this end code is regulating the sequence of the linker and CBD Chaetomium thermophilum Cel7A was synthesized by PCR, using the following primers:

5'-TAAACATATGTTATCTACTCCAACATCAAGGTCGGACCCATCGGC-TCGACCGTCCCTGGCCTTGAC-3' (forward sequence)

and

5'-TATATGCGGCCGCAAGCTTTACCATCAAGTTACTCCAGCAAATCA-GGGAACTG-3' (reverse sequence).

PCR reaction mixture contained 1 × DyNAzymeEXT-reaction buffer (Finnzyme, Finland), 15 mm Mg2+, 0.2 mm dNTPs, 2 µm of each primer, 0.6 units of DyNAzymeEXT DNA polymerase (Finnzyme, Finland) and approximately 75 ng/30 ál of DNA templates containing cel7A gene full length from Chaetomium thermophilum. Conditions for PCR reactions were as follows: 2 min initial denaturation at 98aboutC, then 30 cycles of 30 sec at 98aboutC, 30 sec annealing at 68about(Difference ±4aboutC), and elongation for 30 sec at 72aboutC and final elongation at 72aboutC for 10 min. Specific DNA fragment in a PCR reaction was obtained when the annealing temperature in the range from 64 to 68.5aboutC. Synthesized CBD-fragment Chaetomium thermophilum was Legerova after Thermoascus aurantiacus cel7A gene that made the connection point GPIGST between domains. PCR amplificatory fragment in the plasmid was confirmed by sequencing (SEQ ID:29). Designed merged cel7A gene was precisely fused to the T. reesei cbh1 (cel7)-promoter. Termination of transcription was provided by T. reesei cel7A-terminated, and for selection of transformants was used A. nidulans amdS marker gene, as described by Paloheimo et al. (2003).

Linearized Express the traditional cassette was isolated from the vector frame after splitting NotI and transformed into T. reesei A96 motorway-the protoplasts. Transformation was carried out as Penttilä et al. (1987) with modifications described by the Karhunen et al. (1993), making selection with ndimethylacetamide as the sole nitrogen source. Transformants were purified on selective cups through a single conidia to their sporulation on PD.

The production of transformants Thermoascus aurantiacus Cel7A+CBD (SEQ ID: 28 and 30) were analyzed by supernatants crops cultivated in shake flasks (50 ml). Transformants were grown for 7 days in a comprehensive inducing cellulase environment (Joutsjoki et al. (1993), buferiruemoi 5% KH2PO4at a pH of 5.5. Cellobiohydrolase activity was analyzed using 4-methylumbelliferyl-β-D-lactoside (MUL) substrate in accordance with van Tilbeurgh et al., 1988. The genotypes of the selected transformants was confirmed using Southern-blots, in which was included a few genomic hydrolization, and appropriate expression cassette was used as a probe. SDS-PAGE analysis showed that the recombinant enzymes Thermoascus aurantiacus Cel7A+CBD was producirovanie as a stable fusion proteins in T. reesei.

The selected transformant producing Ta Cel7+Tr CBD-fusion protein (SEQ ID: 28), also cultivated in 2 l bioreactor at the 28aboutWith the above environment within 3-4 days with pH control, 4,4±0,2 (NH3/H3PO4to get material for tests note the wetlands. Supernatant recovered by centrifugation and filtration through a Seitz-150 K and EK-filters (Pall SeitzSchenk Filtersystem GmbH, Bad Kreuznach, Germany).

Example 16. Comparison of the constants of the Michaelis-Menten and inhibition cellobiose purified recombinant cellobiohydrolase

Constants of the Michaelis-Menten and inhibition cellobiose was determined by cellobiohydrolases, heterologic produced in T. reesei (Examples 14 and 15). The enzymes were purified as described in Example 2. The concentration of protein in the purified enzymes was measured by its absorption at 280 nm using a theoretical molar extinction coefficient, which was calculated based on amino acid sequences (Gill and von Hippel, 1989).

Kinetic constants (the values of Km and kcat) and the inhibition constant cellobiose (Ki) for Tr CBHI/Cel7A, Ta CBH/Cel7A, At CBH/Cel7A and Ct CBH/Cel7A was measured using CNPLac (2-chloro-4-nitrophenyl-β-D-lactose) as a substrate at ambient temperature (22aboutC) in 50 mm sodium phosphate buffer, pH of 5.7. To determine the inhibition constants (Ki) were measured eight different concentrations of the substrate (31-4000 μm) in the presence of a range of five concentrations of inhibitor (0-100 μm or 0-400 μm), where the bracket is Ki. All experiments were placed in microtiter plates, and the total reaction volume was 200 μl. The initial velocity in each case was measured by continuous MES is by monitoring the release of chloro-nitrophenolate ion (CNP, 2-chloro-4-nitrophenolate) by means of measurements at 405 nm using a microtiter tablet reader Varioscan (Thermolabsystem). The results were calculated by standard curve SNP (from 0 to 100 μm). Used concentrations of the enzymes were: Tr CBHI/Cel7A 2,46 μm, Ta CBH/Cel7A was 1.58 μm, Ct CBH/Cel7A of 0.79 μm and At CBH/Cel7A 3 μm. Constants Km and kcat were calculated from the appropriate equation of Michaelis-Menten using the program Origin. To distinguish between competitive and mixed inhibition and determination of inhibition constants (Ki) used charts Leinweber-Burke, opposite charts (relatively LWB-slope [GIc2; cellobiose]) and charts Haines.

The results of kinetic measurements are shown in Table 12 and 13. As you can see, Ct CBH/Cel7A clearly has a higher number of revolutions (kcat) for CNPLac, as well as higher specificity constant (kcat/Km) compared to the CBHI/Cel7A of T. reesei. Cellobiose (Glc2) is a competitive inhibitor for all measured cellulases, and Tr CBHI/Cel7A (used as control) most strongly inhibited (i.e. had the lowest value of Ki) cellobiose. At CBHI/Cel7A had more than 7 times higher inhibition constant compared to that of Tr CBHI/Cel7A. These results indicate that all three new cellobiohydrolase could work better in the cellulose hydrolysis due to reduced cellobiose Engibarov the Yu compared with cellobiohydrolase I Trichoderma reesei Cel7A.

Table 12
Comparison constants cellobiose inhibition of four GH family 7 cellobiohydrolase measured on CNPLac in 50 mm sodium phosphate buffer, pH 5,7, 22about
EnzymeKi (μm)The type of inhibition
Ct Cel7A39competitive
Ta Cel7A107competitive
At Cel7A141competitive
Tr Cel7A19competitive

Table 13
Comparison of the kinetic constants of the Michaelis-Menten cellobiohydrolase Cel7A Chaetomium thermophilum and CBHI/Cel7A of T. reesei measured on CNPLac in 50 mm sodium phosphate buffer, pH 5,7, 22about
Enzymekcat (min-1)Km (µm)ncat/Km ((min-1 M -1)
Ct Cel7A18,819609,5 103
Tr Cel7A2,65205,0 103

Example 17. The hydrolysis of crystalline cellulose (Avicel) recombinant cellobiohydrolase

Purified recombinant cellobiohydrolase Ct Cel7A, Ta Cel7A, Ta Cel7A+Tr CBD, Ta Cel7A+Ct CBD, At Cel7A, as well as a core option Ct Cel7A (see below) were tested in equimolar amounts in the hydrolysis of crystalline cellulose at two temperatures, 45aboutWith the 70aboutC; cleaned Tr Cel7A of T. reesei and its core option (see below) were used for comparison. Analysis of the hydrolysis of crystalline cellulose (Ph 101, Avicel; Fluka, Switzerland) was performed in a 1.5 ml tube 50 mm sodium acetate, pH 5.0. Avicel was shaken at 45 or 70aboutWith enzyme solution (1.4 µm), and the final volume of the reaction mixture was 325 ál. After hydrolysis for 24 hours was followed by sampling at six different time points and the reaction was stopped by adding 163 μl stop reagent containing 9 volumes of ethanol and 1 volume of 1 M glycine (pH 11). The solution was filtered through Millex GV 13 0.22 μm filter unit (Millipore, Billerica, MA, USA). The formation of soluble reducing sugars in the supernatant was determined by pair-hydroxybenzeneacetic (PAHBAH) method (Lever, 1972), using standard cellobiose curve (50-1600 μm cellobiose). In freshly prepared 0.1 to PAHBAH (Sigma-Aldrich, St. Lous, Mo, USA) in 0.5 M NaOH (100 μl) solution was added 150 μl of filtered sample and boiled for 10 minutes, after which the solution was cooled on ice. The absorption of the samples was measured at 405 nm.

Crust options cellobiohydrolase containing CBD in their native form, was prepared as follows: Ct Cel7A and Tr Cel7A were subjected to proteolytic cleavage to remove the pulp-binding domain. Cleavage by papain (Papaya Latex, 14 U/mg, Sigma) native cellobiohydrolase performed at 37aboutC for 24 h in a reaction mixture composed of 10 mm L-cysteine and 2 mm EDTA in 50 mm sodium acetate buffer (pH 5.0) with the addition of papain (tested two concentrations of papain: one-fifth and one-tenth the number of papain from the total number of Cel7A in the reaction mixture). The obtained core protein was purified using DEAE Sepharose FF (Farmacia, Uppsala, Sweden) anion-exchange column as described above. The product was analyzed on SDS-PAGE.

The results of hydrolysis at 45aboutWith the 70aboutShown in the Figure 4 and 5, respectively. The results clearly show that all cellobiohydrolase find a more rapid and complete hydrolysis at both temperatures compared to cellobiohydrolase Cel7A of T. reesei prior art. If 70aboutWith thermostable CE is abiogenous from Thermoascus aurantiacus ALKO4242 and Chaetomium thermophilum ALKO4265 surpass T. reesei Cel7A and when Thermoascus Cel7A-cor coupled with the CBD of T. reesei Cel7A (Ta Cel7A+Tr CBD). It was amazing that cellobiohydrolase, isolated and cloned in this study, surpass, when contain CBD, the speed and formation of the product in the hydrolysis of crystalline cellulose cellobiohydrolase Cel7A of T. reesei (CBD) of the prior art in the traditional temperature hydrolysis 45aboutWith the same concentration of enzyme. These results are also consistent with those of the enzyme preparations (At Cel7A and Ct Cel7A), which were purified from the original masters and tested in Avicel-hydrolysis (50aboutC, 24 h) (Example 2, table 1).

Example 18. Cloning endoglucanase gene from Acremonium thermophilum ALKO4245, Chaetomium thermophilum ALKO4261 and Thermoascus aurantiacus ALKO4242

Used standard methods of molecular biology as described in Example 13. Construction of genomic libraries Acremonium, Chaetomium, and Thermoascus were described in Example 12.

Peptides coming from the purified endoglucanases Acremonium and Chaetomium, had homology with several endoglucanase family 45 glycosylglycerols, such as endoglucanases Cel45A Melanocarpus albomyces (AJ515703) and endoglucanases Humicola insolens (A35275), respectively. Peptides came from endoglucanase Thermoascus, had almost 100%identity with the published sequence of endoglucanase EG1 Thermoascus aurantiacus (AF487830). In order to amplify the probe to the TFR is mapping genomic libraries Acremonium and Chaetomium, based on the peptide sequences were designed degenerate primers. The order of the peptides in the protein sequences and the corresponding sense and antisense nature of the primers deduced from comparison with homologous published endoglucanase. Primernye sequence and the corresponding peptides are listed in Table 14. Thanks to the almost 100%identical peptides Thermoascus with the published sequence endoglucanase gene PCR amplified directly from genomic DNA.

Table 14
The oligonucleotides were synthesized and used as PCR primers for the amplification of a probe for screening genes Acremonium thermophilum cel45A (EG_40) and Chaetomium thermophilum cel7B (EG_54) from the corresponding genomic libraries
ProteinPeptideThe position of the primers(aPrimernye sequence(b
At EG_40Peptide 51-6TAYTGGGAYTGYTGYAARCC
WFQNADN(c RTTRTCNGCRTTYTGRAACCA
Ct EG_54Peptide 73-7GCAAGCTTCGRCARAARTCRTCRTT(d
Peptide 25-9GGAATTCGAYCARACNGARCARTA(e
(a - Amino acid peptide that was used to construct primerno sequence;
(b - N=A, C, G or T; R=A or G; Y=C or T;
(c - Peptide originating not from purified protein EG_40 Acremonium, and the sequence (AJ515703) Cel45A M. albomyces homologous EG_40;
(d - 5'-end of the oligonucleotide was added restriction site a HindIII;
(e - to 5'-end of the oligonucleotide was added restriction site An EcoRI.

Specific probe to gene cel45A Acremonium thermophilum for screening genomic libraries are amplified using forward (TAYTGGGAYTGYTGYAARCC) and reverse (RTTRTCNGCRTTYTGRAACCA) primers using genomic DNA as template. PCR reaction mixture contained 50 mm Tris-HCl, pH of 9.0, 15 mm (NH4)2SO4, 0,1% Triton X-100, 1.5 mm MgCl2, 0.1 mm dNTPs, 0.5 μg of each primer, 1 unit of Dynazyme EXT DNA polymerase (Finnzymes, Finland) and approximately 0.5 μg of genomic DNA of Acremonium. Conditions for PCR reactions were as follows: 5 min initial denaturation at 95aboutC, then 30 cycles of 1 min at 95about, Min annealing at 50-60 aboutC, 2 min elongation at 72aboutC and final elongation at 72aboutC for 10 min For amplification specific probe to cel7B-gene Chaetomium thermophilum (codereuse Ct EG_54) used direct primer GGAATTCGAYCARACNGARCARTA and reverse primer GCAAGCTTCGRCARAARTCRTCRTT. PCR reaction mixture contained 10 mm Tris-HCl, pH 8,8, 50 mm KCl, 0.1% of Triton X-100, 1.5 mm MgCl2, 0.2 mm dNTPs, 250 pmol each primer, 2 units of Dynazyme II DNA polymerase (Finnzymes, Finland) and approximately 2 µg of genomic DNA Chaetomium. Conditions for PCR reactions were as described above except that the annealing was carried out at 45-50aboutC.

From PCR reactions Acremonium were obtained two products. DNA fragments of about 0.6 and 0.8 KBP KBP was isolated from agarose gel and cloned into pCR4-TOPO® TA vector (Invitrogen, USA), obtaining the plasmid pALK1710 and pALK1711, respectively. DNA products were characterized by sequencing and performing Southern blot hybridization with genomic DNA of Acremonium, split multiple restriction enzymes. Drawings hybridization obtained for the two fragments in simple terms flushing, suggest that genomic library Acremonium could be skanirovaniya two alleged endoglucanase gene. The deduced amino acid sequence of both PCR products have homology with several published endoglucanase sequences glycosylglycerols families who tion 45 (BLAST program, National Center for Biotechnology Information; Altschul et al., 1990).

One PCR product of the expected size (estimated from homologous endoglucanases sequence Humicola insolens, A35275), was obtained from a PCR reaction Chaetomium. A DNA fragment of about 0.7 KBP were cloned in pCR4-TOPO® TA vector (Invitrogen, USA) to obtain plasmid pALK2005 and analyzed as described above. Deduced amino acid sequence of the PCR product had homology with several published cellulase sequences glycosylglycerols family 7 (BLAST program, version 2.2.9 in NCBI, the national Center for Biotechnology Information; Altschul et al., 1990).

Insert of the plasmid pALK1710, pALK1711 and pALK2005 allocated by splitting restrictionenzyme enzymes and were marked by digoxigenin in accordance with the supplier's instructions (Roche, Germany). Was skanirovali plaque size of about 1-2×105from amplified genomic library Acremonium or Chaetomium. The hybridization temperature was 68aboutC and the filters were washed 2 x 5 min at RT using 2 × SSC and 0.1% SDS, and then 2×15 min at 68aboutWith using of 0.1 × SSC and 0.1% SDS. There have been several positive plaques from each screening was cleared five or six heavily gibridizatsiya plaques. Phage DNA was isolated and analyzed by Southern blot hybridization. Restriction fragments, hybrids the present probe was subcloned into the vector pBluescript II KS+(Stratagene, USA), and the relevant part of the sequence. In all cases subcloned phage fragment contained a gene of interest full length. Table 15 gives a summary of the probes used for screening endoglucanase genes, phage clones from which these genes were selected, the selected restriction fragments containing the genes for the complete length with their promoter and termination regions, the names of the plasmids containing subcloned phage fragment, and custody of the rooms in the German Collection of Microorganisms and Cell Cultures (DSM) for E. coli strains carrying these plasmids.

Table 15
The probes used for cloning endoglucanase gene, phage clone selected subclan, the name of the plasmid and the corresponding Deposit number of strains of E.coli
GeneGenomic libraryThe probe used for screeningPhage cloneSubcloned fragmentPlasmidDepository number of E.coli
At cel45A A. thermophilum ALKO4245pALK1710P24the 5.5 KBP SmaIpALK1908DSM 17324
At cel45BA. thermophilum ALKO4245pALK1711P41the 6.0 KBP XhoIpALK1904DSM 17323
Ct cel7BC. thermophilum ALKO4261pALK2005P55a 5.1 KBP BamHIpALK2010DSM 17729

Cel5A gene Thermoascus aurantiacus (encoding EG_28) (SEQ ID NO: 9) amplified directly from selected genomic DNA by PCR reaction. Direct (ATTAACCGCGGACTGCGCATCATGAAGCTCGGCTCTCTCGTGCTC) and reverse (AACTGAGGCATAGAAACTGACGTCATATT) primers were used for amplification were designed based on the published gene eg1 T. aurantiacus (AF487830). PCR reaction mixture contained 1 × Phusion HF buffer, 0.3 mm dNTPs, 0.5 µm each primer, 2 units PhusionTM DNA polymerase (Finnzymes, Finland) and approximately 0.25 μg genomic DNA Thermoascus. Conditions for PCR reactions were as follows: 5 min initial denaturation at 95aboutC, then 25 cycles of 30 s at 95aboutC, 30 s annealing at 57-67aboutWith a 2.5 min elongation at 72aboutAnd the conclusion is positive elongation at 72 aboutC for 5 minutes Amplificatory product of 1.3 KBP containing the exact gene (from START to STOP codon)was cloned as a SacII- > PST fragment into the vector pBluescript II KS+. Two independent clones sequenced and one clone was selected and designated as pALK1926. Depository number of strains of E.coli containing pALK1926 in the German Collection of Microorganisms and Cell Cultures - DSM 17326.

Relevant information about genes and deduced protein sequences (SEQ ID nos: 9-16) are given in Table 16 and 17, respectively. The peptide sequence of the purified endoglucanases Acremonium EG_40 (gene At cel45A), Chaetomium EG_54 (gene Ct cel7B), and Thermoascus EG_28 (Ta gene cel5A) were detected in the corresponding deduced amino acid sequences of the cloned genes, confirming that were cloned genes.

Table 16
Summary data for endoglucanase genes isolated from Acremonium thermophilum, Chaetomium thermophilum, and Thermoascus aurantiacus
Endoglucanase geneThe length of the introns (BP)(aThe coding region (BP)(bThe number of intronsThe length of the intron (BP)SEQ ID NO:
At cel45A1076891259, 12311
At cel45B10137532155, 10213
Ct cel7B12781275--15
Ta cel5A13171005555, 60, 59, 74, 619
(a STOP codon is included;
(b - a STOP codon is not included.

Table 17
Summary data on these endoglucanase sequences of Acremonium thermophilum, Chaetomium thermophilum, and Thermoascus aurantiacus. ss, signal sequence
Endoglucanase proteinQty AK th.Length ss NN/HMM(aCBD(bThe predicted MV (Yes, ss is not on) (withThe predicted pI (ss excl.)The alleged sites of N-glycosylation(dSEQ ID NO:
At EG_4029721/21Yes, K265-L297286254,79212
At EG_40_25120/20No239726,11214
Ct EG_5442517/17No453585,44116
Ta EG_2833530(eNo337124,30110
(a Prediction of the signal sequence was made using the program SignalP V3.0 (Nielsen et al., 1997; Bendten et al., 2004); the value of NN was obtained, and the uses of the neural network, and the value of the HMM - using hidden modules Markov;
(b - Presence of pulp-binding domain; specified amino acids C-terminal CBD (numbering according to the full length polypeptide);
(c - Predicted signal sequence is not included. The prediction was made using the Compute pI/MW on the ExPASy server (Gasteiger et al., 2003);
(d - Proposed sites of N-glycosylation sites N-X-S/T were predicted using the NetNGlyc 1.0 in (Gupta et al., 2004);
(e - According to Hong et al., 2003a.

The deduced protein sequence of endoglucanases Acremonium EG_40 (At cel45A) and EG_40_ (At cel45B), Chaetomium EG_54 (Ct cel7B), and Thermoascus EG_28 (Ta cel5A) have homology with cellulases of glycosylglycerols family 45 (Acremonium), family 7 (Chaetomium) and family 5 (Thermoascus), so the selected genes are identified as members of these gene families. The closest homologues of endoglucanases EG_40/Cel45A and EG_40_/Cel45B Acremonium are endoglucanase Thielavia terrestris (CQ827970, 77,3% identity) and Myceliophthora thermophila (AR094305, 66,9% identity), respectively (table 18). Two dedicated endoglucanase family 45 Acremonium identical to one another only by 53.7%. Of these enzymes only EG_40/Cel45A contains pulp-binding domain (CBD).

The closest homology to the predicted protein sequence endoglucanase EG_54/Cel7 In Chaetomium found in cellulase sequence (AJ55704) Cel7A Melanocarpus albomyces. Identity between two protein sequences is 70,6%.

Protein sequence selected endoglucanase Thermoascus aurantiacus completely identical to the published sequence EGI T. aurantiacus (AF487830, table 18). The closest homology was detected in the sequence of β-glucanase Talaromyces emersonii (AX254752, 71,1% identity).

Table 18
Comparison of derived endoglucanases EG_40, EG_40_/Cel45B Acremonium thermophilum, EG_54/Cel7 In Chaetomium thermophilum and EG_28/Cel5A Thermoascus aurantiacus with their homologous counterparts. The alignment was performed using the Needle program of the EMBOSS software package. *indicates endoglucanase encoded by a gene cloned in this study
The body, the enzyme and the access numberIdentity %
Acremonium thermophilum EG_40100,0
Thielavia terrestris EG45, CQ82797077,3
Melanocarpus albomyces Cel45A, AJ51570375,3
Neurospora crassa hypothetical XM_32447768,9
Humicola grisea var thermoidea, EGL3, AB003107 67,5
Humicola insolens EG5, A2363567,3
Myceliophthora thermophila SEM. 45, AR09430557,9
* Acremonium thermophilum EG_40_53,7
Acremonium thermophilum EG_40_100,0
Myceliophthora thermophila SEM. 45, AR09430566,9
Magnaporthe grisea 70-15 hypothetical, XM_36340261,9
Thielavia terrestris EG45, CQ82797056,8
* Acremonium thermophilum EG_4053,7
Melanocarpus albomyces Cel45A, AJ51570352,8
Chaetomium thermophilum EG_54100,0
Melanocarpus albomyces Cel7A, AJ51570470,6
Humicola grisea var thermoidea, EGI, D6351668,8
Humicola insolens EGI, AR01224467,7
Myceliophthora thermophila EGI, AR07193461,7
Fusarium oxysporum var lycopercisi EGI, AF2921053,5
Fusarium oxysporum EGI, AR012243 52,6
Thermoascus aurantiacus EG_28100,0
Thermoascus aurantiacus EG, AX812161100,0
Thermoascus aurantiacus EGI, AY05512199,4
Talaromyces emersonii β-glucanase, AX25475271,1
Talaromyces emersonii EG, AF44000370,4
Aspergillus niger EG, A6966370,1
Aspergillus niger EG, A6244169,9
Aspergillus niger EG, AF33151869,6
Aspergillus aculeatus EGV, AF05451268,5

Example 19. Production of recombinant endoglucanases in Trichoderma reesei

For producing recombinant proteins Acremonium EG_40/Cel45A and EG_40_/Cel45B and Thermoascus EG_28/Cel5A were constructed expression plasmids as described in Example 14. The linearized expression cassette (table 19) was isolated from the vector frame by cleavage with restriction enzymes, and transformed into T. reesei A96 motorway and transformants were purified as described in Example 14.

Table 19
The endoglucanasesExpression plasmidThe size of the expression cassette(aHeterologous terminator(b
At EG_40pALK1920the 10.9 KBP NotI156 BP (HindIII)
At EG_40_pALK1921the 8.6 KBP EcoRI282 BP (SspI)
Ta EG_28pALK1930the 8.6 KBP NotINo
(a - Expression cassette for transformation of T. reesei was isolated from vector frame by splitting EcoRI or NotI;
(b - indicates the number of nucleotides after the STOP codon in the cloned gene, which are included in the expression cassette. In brackets restriction site in the 3'region of the gene, which was used in constructing expression cassettes.

Production endog the aluminum maker of the transformants were analyzed by supernatant cultures, cultivated in shake flasks (50 ml). Transformants were grown as in Example 14, and the enzymatic activity of the recombinant protein was measured in the supernatant of the culture, as the release of reducing sugars from carboxymethyl cellulose (2% (W/V) CMC) at the 50aboutWith 50 mm citrate buffer, pH of 4.8, essentially as described by Bailey and Nevalainen, 1981; Haakana et al., 2004. The production of recombinante proteins was determined by supernatant cultures by SDS-polyacrylamide gel electrophoresis. Acremonium EG_40-specific polyclonal antibodies were obtained in rabbits (University of Helsinki, Finland). The expression EG_40 was checked by Western blot analysis using anti - EG_40 antibodies using the ProtoBlot Western blot AP (Promega). The genotypes of the selected transformants were analyzed by Southern-blotting, using as a probe expression cassette.

pH-optimum heterologic endoglucanases produced was determined in a universal buffer of MacLaine in the range of pH of 4.0 to 8.0, using carboxymethylcellulose as a substrate. As shown in Figure 6A, the wide range of pH (4,-6,0) refers to the protein EG_40/Cel45A Acremonium with an optimum at pH 5.5. the pH-Optima for other heterologic produced endoglucanases amount of 5.0-5.5 and 6.0 for Acremonium EG_40_/Cel45B and Thermoascus EG_28/Cel5A, respectively. The optimum temperature for enzymatic activity of these endoglucanases was determined in the temperature range of 50-85 aboutSince, as described above. It was determined that the highest enzyme activity is manifested at 75, 60 and 75aboutFor Acremonium EG_40/Cel45A, EG_40_/Cel45B and Thermoascus EG_28/Cel5A, respectively (Figure 6B).

Selected transformants were cultured as described in Example 14 2-liter bioreactor for four days (the 28aboutWith a pH of 4.2)to obtain material for tests on the application.

Example 20. Cloning of beta-glucosidase genes Acremonium thermophilum ALKO4245, Chaetomium thermophilum ALKO4261 and Thermoascus aurantiacus ALKO4242

Used standard methods of molecular biology as described in Example 13. Construction of genomic libraries Acremonium, Chaetomium, and Thermoascus were described in Example 12.

Peptides derived from purified β-glucosidase Acremonium, Chaetomium, and Thermoascus, had homology with several β-glucosidase of glycosylglycerols family of 3, such as β-glucosidase Acremonium cellulolyticus (BD168028), Ttichoderma viride (AY368687) and Talaromyces emersonii (AY072918), respectively. For amplification of a probe for screening genomic libraries Acremonium, Chaetomium, and Thermoascus based on the peptide sequences were designed degenerate primers. The order of the peptides in the protein sequence and the corresponding sense or antisense nature of the primers deduced from comparison with homologous published β-glucosidase. Primernye sequence and the corresponding PEP the IDA are listed in Table 20.

td align="left"> 2-7
Table 20
The oligonucleotides were synthesized and used as PCR primers for amplificatoare probe for screening genes cel3A (βG_101) Acremonium thermophilum, cel3A (βG_76) Chaetomium thermophilum and cel3A (βG_81) Thermoascus aurantiacus from the corresponding genomic libraries
ProteinPeptideThe position of the primer(aPrimera sequence(b
At βG_101EKVNLT(cGARAARGTNAAYCTNAC
Peptide 46-11YTTRCCRTTRTTSGGRGTRTA
Ct βG_76Peptide 64-9TNTGYCTNCARGAYGG
Peptide 13-8TCRAARTGSCGRTARTCRATRAASAG
Ta βG_81Peptide 31-5AARGGYGTSGAYGTSCAR
Peptide 1YTTRCCCCASGTRAASGG
(a - Amino acid peptide that was used to construct primerno sequence;
(b To decrease the degeneracy some codons were selected according to the preferences of fungi. N=A, C, G or T; R=A or G; S=C or G; Y=C or T;
(c - Peptides originating not from purified protein βG_101 Acremonium, and β-glucosidase sequence A. cellulolyticus (BD168028), homologous βG_101.

Probes for screening the constructed genomic libraries amplified with combinations of the above primers (table 20), using genomic DNA of Acremonium, Chaetomium, and Thermoascus as matrices. PCR reaction mixture contained 50 mm Tris-HCl, pH of 9.0, 15 mm (NH4)2SO4, 0,1% Triton X-100, 1.5 mm MgCl2, 0.1-0.2 mm dNTPs, and 0.25 μg of each primer, 1 unit of Dynazyme EXT DNA polymerase (Finnzymes, Finland) and priblisitelno 0.5 μg of genomic DNA. Conditions for PCR reactions were as follows: 5 min initial denaturation at 95aboutC, then 30 cycles of 1 min at 95aboutC, 1 min annealing at 40about(Acremonium DNA as template), when 50about(DNA Chaetomium as a matrix) or 63about(DNA Thermoascus as a matrix), 2-3 min elongation at 72aboutC and final elongation at 72aboutC for 5-10 minutes

Specifications the ical PCR products of the expected size (estimated by homologous β-glucosidase sequences BD168028, AY072918 and AY368687) was isolated from an agarose gel. DNA fragments of a size of about 1.8 KBP (Acremonium), a 1.5 KBP (Chaetomium) and 1,52 KBP (Thermoascus) cloned into the vector pCR4-TOPO®TA (Invitrogen, USA) to obtain plasmid pALK1924, pALK1935 and pALK1713, respectively. DNA products were characterized by sequencing and holding Southern blot hybridization with genomic DNA cleaved with several restriction enzymes. The patterns of hybridization in simple terms flushing suggest that from a genomic library Acremonium, Chaetomium, and Thermoascus could be allocated one estimated β-glucosidase gene. Deduced amino acid sequences of all three PCR products have homology with several published β-glucosidase sequences glycosylglycerols family 3 (BLAST program, national Center for Biotechnology Information; Altschul et al., 1990).

Insert of the plasmid pALK1924, pALK1935 and pALK1713 was isolated by cleavage with restriction enzymes and were marked by digoxigenin in accordance with the supplier's instructions (Roche, Germany). Approximately 1-2×105plaques from genomic libraries Acremonium, Chaetomium, and Thermoascus was skanirovali as described in Example 18. There have been several positive plaques from each screening was purified five or six heavily gibridizatsiya. Phage DNA was isolated and analyzed by Southern blot hybridization. estriction fragments, hybridities probe was subcloned into the vector pBluescript II KS+(Stratagene, USA) and the relevant part of the sequence. In all cases subcloned phage fragment contained a subject of interest gene full length. Table 21 summarizes the data for the probes used for screening β-glucosidase genes rahovym clones, which were selected genes selected restriction fragments containing the genes for the complete length with their promoter and termination regions, the names of the plasmids containing subcloned phage fragment and Depository rooms at German Collection of Microorganisms and Cell Cultures (DSM) of E. coli strains carrying these plasmids.

Relevant information about genes and deduced protein sequences (SEQ ID nos: 21-26) are summarized in Table 22 and 23, respectively. Peptide posterolateral purified proteins Acremonium βG_101 (At Cel3A), Chaetomium βG_76 (Ct Cel3A) and Thermoascus βG_81 (Ta Cel3A) were detected in the corresponding deduced amino acid sequences of the cloned genes, confirming that were cloned genes.

Table 22
Summary data for β-glucosidase genes isolated from Acremonium thermophilum, Chaetomiu thermophilum and Thermoascus aurantiacus
β-glucosidase geneThe length of the introns (BP)(aThe coding region (BP)(bThe number of intronsThe length of introns (BP)SEQ ID NO:
At cel3A28212583392, 74, 6923
Ct cel3A2257220215225
Ta cel3A308425297134, 67, 56, 64, 59, 110, 6221
(a STOP codon is included.
(b - a STOP codon is not included.

Table 23
Totals derived β-glucosidase sequences Thermoascus aurantiacus, Chaetomium thermophilum and Acremonium thermophilum. ss, signal sequence
β-glucosidase proteinQuantity am the NOC. Length ss NN/HMM(aC-terminal CBD(bThe predicted MW (Da, ss excl.)(cThe predicted pI (ss excl.)The alleged sites of N-glycosylation(dSEQ ID NO:
At βG_10186119/18No914345,46824
Ct βG_7673420/20No764576,3226
Ta βG_8164319/19No899244,95822
(a Prediction of the signal sequence was made using the program SignalP V3.0 (Nielsen et al., 1997; Bendtsen et al., 2004); the value of NN was obtained using the neural circuit, and the value of the HMM - using hidden modules Markov.
(b - Presence of pulp and the binding is found domain protein;
(c - Predicted signal sequence is not included. The prediction was made using the Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003);
(d - Proposed sites of N-glycosylation sites N-X-S/T were predicted using the NetNGlyc 1.0 in (Gupta et al., 2004).

The deduced protein sequence of β-glucosidase Acremonium βG_101/Cel3A, Chaetomium βG_76/Cel3A, and Thermoascus βG_81/Cel3A have homology with enzymes glycosylceramide family of 3, identifying, thus belonging selected genes in this gene family. The closest analogues of β-glucosidase Acremonium, Chaetomium, and Thermoascus are those of Magnaporthe grisea (β-glucosidase, AY849670), Neurospora crassa (hypothetical, XM_324308) and Talaromyces emersonii (β-glucosidase, AY072918), respectively (table 24). The highest detected identity of the sequences (73,2%) had C. thermophilum βG_76/Cel3A with a hypothetical protein from N. Crassa, which confirmed that new genes have been cloned.

Table 24
Comparison of the deduced β-glucosidase βG_101/Cel3A Acremonium thermophilum, βG_76/Cel3A Chaetomium thermophilum and βG_81/Cel3A Thermoascus aurantiacus with their homologous counterparts. The alignment was performed using the Needle program of the EMBOSS software package. *indicates endoglucanase encoded by the gene, cloned in the data is m research

The body, the enzyme and the access numberIdentity %
* Acremonium thermophilum βG_101100,0
Magnaporthe grisea β-glucosidase, AY84967073,1
Neurospora crassa hypothetical XM_33087171,1
Trichoderma reesei Cel3B, AY28137465,2
* Thermoascus aurantiacus βG_8162,2
Aspergillus aculeatus β-glucosidase, D6408859,5
Talaromyces emersonii β-glucosidase, AY072918of 58.9
Aspergillus oryzae, AX61673858,2
Acremonium cellulolyticus β-glucosidase, BD16802857,2
* Chaetomium thermophilum βG_76of 40.9
Chaetomium thermophilum βG_76100,0
Neurospora crassa hypothetical XM_32430876,9
Magnaporthe grisea 70-15 hypothetical, XM_364573to 70.2
Trichoderma viridae BGI, AY36868765,8
Acremonium cellulolyticus β-glucosidase, BD16802841,2
* Acremonium thermophilum βG_101of 40.9
Trichoderma reesei Cel3B, AY28137440,0
* Thermoascus aurantiacus βG_8139,9
* Thermoascus aurantiacus βG_81100,0
Talaromyces emersonii β-glucosidase, AY07291873,2
Aspergillus oryzae, AX61673869,5
Aspergillus aculeatus β-glucosidase, D6408868,0
Acremonium cellulolyticus β-glucosidase, BD16802865,7
* Acremonium thermophilum βG_10162,2
Trichoderma reesei Cel3B, AY28137457,9
* Chaetomium thermophilum βG_7639,9

Example 21. Production of recombinant beta-glucosidase in Trichoderma reesei

For producing recombinant proteins Acremonium βG_101/Cel3A, Chaetomium βG_76/Cel3A, and Thermoascus βG_81/Cel3A were constructed expression plasmids as described in Example 14. Linearized cressionnie cassettes (table 25) was isolated from the vector frame by cleavage with restriction enzymes, transformed into T. reesei A96 motorway or A33 (in both strains deleterows the genes encoding the four major cellulase CBHI/Cel7A, CBHII/Cel6A, EGI/Cel7B and EGII/Cel5A), and transformants were purified as described in Example 14.

Table 25
Expression cassettes designed for producing β-glucosidase βG_101/Cel3A Acremonium thermophilum, βG_76/Cel3A Chaetomium thermophilum and βG_81/Cel3A Thermoascus aurantiacus in Trichoderma reesei. Schematic structure of the expression cassettes described in the Figure 2
β-glucosidaseExpression plasmidThe size of the expression cassette(aHeterologous terminator(b
At βG_101pALK1933the 10.5 KBP NotI300 BP (HindIII)
Ct βG_76pALK200410,1 KBP EcoRI528 BP (XbaI)
Ta βG_81pALK1914the 10.9 KBP EcoRI452 BP (ApoI)
(a - Expression cassette for transformation of T. reesei was allocated from, etc) is REGO frame by splitting EcoRI or NotI;
(b - indicates the number of nucleotides after the STOP codon in the cloned gene, which are included in the expression cassette. In brackets restriction site in the 3'region of the gene, which was used in constructing expression cassettes.

The production of β-glucosidase the transformants were analyzed by supernatants crops cultivated in shake flasks (50 ml). Transformants were grown as in Example 14, and the enzymatic activity of the recombinant protein was measured in the culture supernatant using 4-nitrophenyl-β-D-glucopyranoside substrate, as described by Bailey and Nevalainen, 1981. The production of recombinante proteins was determined by supernatant cultures by SDS-polyacrylamide gel electrophoresis. In addition, the expression of Thermoascus βG_81 was checked by Western blot analysis using anti - βG_81 antibodies as described in Example 19. The genotypes of the selected transformants were analyzed by Southern-blotting, using as a probe expression cassette.

pH-optimum heterologic produced β-glucosidase defined in universal buffer of MacLaine in the range of pH 3.0 to 8.0, using 4-nitrophenyl-β-D-glucopyranoside as substrate. the pH Optima for the Acremonium βG_101, Chaetomium βG_76 and Thermoascus βG_81 be 4.5, and 5.5, and 4.5, respectively (Figure 7A). Optimal the reduction temperature for the enzymatic activity of these β-glucosidase was determined in the temperature range of 50-85 aboutSince, as described above. It was determined that the highest enzyme activity is manifested at 70, 65 and 75aboutFor Acremonium βG_101/Cel3A, Chaetomium βG_76/Cel3A, and Thermoascus βG_81/Cel3A, respectively (Figure 7B).

Selected transformants were cultured as described in Example 14 2-liter bioreactor for four days (the 28aboutWith a pH of 4.2)to obtain material for tests on the application.

Example 22. Cloning xylanase genes Acremonium thermophilum ALKO4245 and Thermoascus aurantiacus ALKO4242

Used standard methods of molecular biology as described in Example 13. Construction of genomic libraries Acremonium was described in Example 12.

Peptides derived from the purified xylanase Acremonium, had homology with xylanase of glycosylglycerols family of 10, such as Humicola grisea XYNI (AB001030). All peptides originating from xylanase Thermoascus, were fully identichny with the published sequence XYNI Thermoascus aurantiacus (AJ132635), identifying thus the purified protein as the same enzyme. Thanks xylanases gene Thermoascus was amplified by PCR from genomic DNA.

For amplification of a probe for screening xylanase gene from Acremonium genomic library based on the peptide sequences were designed degenerate primers (Example 11, table 5). The order of the peptides in the protein sequence and is suitable the sense or antisense nature of the primers deduced from the comparison with the homologous sequence XYNI Humicola insolens (AB001030). Semantic Primera sequence (GAYGGYGAYGCSACYTAYATG) based on Peptide 3 (amino acids 2-8)and antisense primer (YTTYTGRTCRTAYTCSAGRTTRTA) - based Peptide 1 (amino acids 4-11).

The reaction was obtained PCR product of the expected size (estimated by homologous sequences XYNI Humicola insolens AB001030). A DNA fragment of about 0.7 KBP was cloned into the vector pCR4-TOPO®TA (Invitrogen, USA), obtaining the plasmid pALK1714, and characterized by sequencing. Deduced amino acid sequence of the PCR product had homology with several published xylanase sequences glycosylceramide collection 10 (the BLAST program (national Center for Biotechnology Information; Altschul et al., 1990).

The insert of plasmid pALK1714 was isolated by cleavage with restriction enzymes and were marked by digoxigenin in accordance with the supplier's instructions (Roche, Germany). Approximately 1-2×105plaques from the amplified genomic library was skanirovali as described in Example 18. There have been several positive plaques, five of which strongly gibridizatsiya, was purified. Phage DNA was isolated and analyzed by Southern blot hybridization. Restriction fragment of 3.5 KBP XbaI, hybridities probe was subcloned into the vector pBluescript II KS+(Stratagene, USA), producing plasmid pALK1725. Relevant part pALK1725 sequenced and our and, it contains a gene xyn10A Acremonium thermophilum full length (SEQ ID NO: 19). Depository number of strains of E.coli containing pALK1725 in the German Collection of Microorganisms and Cell Cultures (DSM) 16726.

Gene xyn10A Thermoascus aurantiacus (SEQ ID NO: 17) was amplified directly from selected genomic DNA by PCR reaction. Direct (TTATACCGCGGGAAGCCATGGTTCGACCAACGATCCTAC) and reverse (TTATAGGATCCACCGGTCTATACTCACTGCTGCAGGTCCTG) primers that were used in the gene amplification were designed based on published xynA gene of T. aurantiacus (AJ132635). PCR reaction mixture contained 50 mm Tris-HCl, pH of 9.0, 15 mm (NH4)2SO4, 0,1% Triton X-100, 1.5 mm MgCl2, 0.3 mm dNTPs, 1 μm each primer, 1 unit of Dynazyme EXT DNA polymerase (Finnzymes, Finland) and approximately 0.5 μg of genomic DNA Thermoascus. Conditions for PCR reactions were as follows: 5 min initial denaturation at 95aboutC, then 30 cycles of 1 min at 95aboutC, 1 min annealing at 60-66aboutC, 3 min elongation at 72aboutC and final elongation at 72aboutC for 10 minutes Amplificatory product of 1.9 KBP containing the exact gene (from START to STOP codon)was cloned as ScaII-BamHI fragment into the vector pBluescript II KS+. Were sequenced three independent clones and one clone was selected and designated as pALK1715. Depository number of strains of E.coli containing pALK1715 in the German Collection of Microorganisms and Cell Cultures (DSM) - 16724.

Depository nome the strain E. coli containing pALK1725 in the German Collection of Microorganisms and Cell Cultures (DSM) 16726.

Relevant data about genes and deduced protein sequences (SEQ ID nos: 17-20) are summarized in Table 26 and 27, respectively. The peptide sequence of the purified protein XYN_60 Acremonium and XYN_30 Thermoascus were detected in the corresponding deduced amino acid sequences of the cloned genes (At xyn10A xyn10A and Ta, respectively), confirming that the corresponding genes have been cloned.

Table 26
Summary of xylanase genes isolated from Acremonium thermophilum and Thermoascus aurantiacus
Xylanases geneThe length of the introns (BP)(aThe coding region (BP)(bThe number of intronsThe length of introns (BP)SEQ ID NO:
At xyn10A147112482135, 8519
Ta xyn10A1913987103, 74, 68, 103, 69, 65, 93, 66, 100, 21217
(a STOP codon is included.
(b - a STOP codon is not included.

Table 27
Summary data on these xylanase sequences of Acremonium thermophilum and Thermoascus aurantiacus. ss, signal sequence
Xylanases proteinNumber of aminos.Length ss NN/HMM(aCBD(bThe predicted MW (Da, ss excl.)(cThe predicted pI (ss excl.)The alleged sites of N-glycosylation(dSEQ ID NO:
At XYN_6041619/19Yes, W385-L416425336,321-220
Ta XYN_3032926(eNo32901of 5.81018
(a Prediction of the signal sequence was made using the program SignalP V3.0 (Nielsen et al., 1997; Bendtsen et al., 2004); the value of NN was obtained using the neural circuit, and the value of the HMM - using hidden modules Markov.
(b - Presence of the carbohydrate-binding domain CBD; specified amino acids C-terminal CBD (numbering according to the full length polypeptide);
(c - Predicted signal sequence is not included. The prediction was made using the Compute pI/MW tool at ExPASy server (Gasteiger et al., 2003);
(d - Proposed sites of N-glycosylation sites N-X-S/T were predicted using the NetNGlyc 1.0 in (Gupta et al., 2004);
(e - According Lo Leggio et al., 1999.

The deduced protein sequence of the xylanase Acremonium and Thermoascus have homology with several enzymes glycosylceramide family 10, identifying the relevant genes as members of the family 10 xylanases. The closest detected analog XYN_60/Xyn10A Acremonium is XYLI Humicola grisea (AB001030), showing 67,1%identity with XYN_60 (table 28). The predicted protein sequence selected xylanase XYN_30/Xyn10A Thermoascus aurantiacus completely identical to the sequence published XYNA T. aurantiacus (R, table 28). The highest homology was found in xylanase sequence of Aspergillus niger (A62445, 69,7% identity).

Table 28
Comparison of the deduced xylanases XYN_60/Xyn10A Acremonium thermophilum and XYN_30/Xyn10A Thermoascus aurantiacus with their homologous counterparts. The alignment was performed using the Needle program of the EMBOSS software package. *indicates a xylanase encoded by a gene cloned in this study
The body, the enzyme and the access numberIdentity %
* Thermoascus aurantiacus XYN_30100,0
Thermoascus aurantiacus XynA, P23360100,0
Thermoascus aurantiacus XynA, AF12752999,4
Aspergillus niger xylanase, A6244569,7
Aspergillus aculeatus xylanase, AR13784469,9
Aspergillus terreus SEM. 10 xyn, DQ08743665,0
Aspergillus sojae, XynXI AB04041463,8
Penicilli chrysogenum xylanase, AY58358562,5
* Acremonium thermophilum XYN_ 60100,0
Humicola grisea XYLI, AB00103067,1
agnaporthe grisea 70-15 hypothetical, XM_36494763,8
Aspergillus aculeatus xylanase, AR14983953,7
Talaromyces emersonii xylanase, AX40383151,8
Gibberella zeae xylanase, AY57596251,4
Magnaporthe grisea XYL5, AY14434848,5
Talaromyces emersonii, AX17228746,9

Example 23. Production of recombinant xylanases in Trichoderma reesei

For producing recombinant proteins Acremonium XYN_60/Xyn10A and Thermoascus XYN_30/Xyn10A were constructed expression plasmids as described in Example 14. The linearized expression cassette (table 29) was isolated from the vector frame by cleavage with restriction enzymes, and transformed into T. reesei A96 motorway, and transformants were purified as described in Example 14.

Table 29
The expression cassette designed for the production of xylanase XYN_60/Xyn10A Acremonium thermophilum and XYN_30/Xyn10A Thermoascus aurantiacus in Trichoderma reesei. Schematic structure of the expression cassettes described in the Figure 2
XylanaseExpressi the fair plasmid The size of the expression cassette(aHeterologous terminator(b
At XYN_60pALK19129,0 KBP150 BP (BamHI)
Ta XYN_30pALK1913of 9.3 KBPno
(a - Expression cassette for transformation of T. reesei was isolated from vector frame by EcoRI cleavage;
(b - indicates the number of nucleotides after the STOP codon in the cloned gene, which are included in the expression cassette. In brackets restriction site in the 3'region of the gene, which was used in constructing expression cassettes.

The production of xylanase by the transformants was analyzed by supernatants crops cultivated in shake flasks (50 ml). Transformants were grown as in Example 14, and the enzymatic activity of the recombinant protein was measured in the supernatant of the culture as wywabieni reducing sugars from birch xylan (1% V/V) at 50aboutWith 50 mm citrate buffer, pH of 5.3 as described by Bailey and Poutanen, 1989. Production of recombinant protein was determined in the culture supernatant with p the power of SDS-polyacrylamide gel electrophoresis. In addition, the expression of both xylanases were determined by Western blot analysis using anti-XYN_30 or anti-XYN_60 antibodies as described in Example 19. The genotypes of the selected transformants were analyzed by Southern-blotting, using as a probe expression cassette.

Thermoascus XYN_30/Xyn10A produced in T. reesei, and determine the pH optimum heterologic produced protein in universal buffer of MacLaine in the range of pH 3.0 to 8.0 using birch xylan as substrate (Figure 8A). The optimum pH was determined as 4.5. The temperature optimum for enzymatic activity XYN_30 was defined as the 75about(Figure 8B).

Selected transformants were cultured as described in Example 14, 2-liter bioreactor for four days (the 28aboutWith a pH of 4.2)to obtain material for tests on the application.

Example 24. Effect of recombinant cellobiohydrolase hydrolysis

Effect of purified recombinant cellobiohydrolase has been evaluated in studies of hydrolysis with purified enzymes of T. reesei. The hydrolysis was performed with the control mixtures of purified enzymes in several pre-treated substrates. The filtrates of cultures of T. reesei containing various clones CBH/Cel7A enzymes, obtained as described in Examples 14 and 15, and CBH-enzymes were purified by affinity chromatography as described in Example 2. In addition, the enzyme mixtures used is built of pure cellulase T. reesei (purified as described Suurnäkki et al., 2000). In the experiment used the following cellobiohydrolase:

Thermoascus aurantiacus ALKO4242 CBH (Ta Cel7A)

Thermoascus aurantiacus ALKO4242 CBH (Ta Cel7A) with genetically attached CBD Trichoderma reesei (Ta Cel7A+Tr CBD)

Thermoascus aurantiacus ALKO4242 CBH (Ta Cel7A) with genetically attached CBD Chaetomium thermophilum (Ta Cel7A+Ct CBD)

Acremonium thermophilum ALKO4245 CBH (At Cel7A)

Chaetomium thermophilum ALKO4265 CBH (Ct Cel7A).

Each CBH/Cel7, which was to be tested (dose of 14.5 mg/g dry matter of substrate), used either together with the EGII/Cel5A T. reesei (3.6 mg/g), or in mixtures containing EGI/Cel7B T. reesei (1.8 mg/g), EGII/Cel5A (1.8 mg/g), xylanase pI 9 (Tenkanen et al., 1992) (5000 ncat/g) and acetyl xylan esterase (AXE) (Sundberg and Poutanen, 1991) (250 ncat/g). All mixtures were supplemented with β-glucosidase from a commercial enzyme preparation Novozym 188 (176 ncat/g dry. weight.). In triplicate tubes containing enzyme mixture and 10 mg (dry matter)/ml of substrate, suspended in 0.05 M sodium acetate, incubated while stirring with a magnetic stirrer at 45aboutC for 48 hours to Prepare and control samples with inactivated enzymes and the corresponding substrates. The release of hydrolysis products was measured as reducing sugars by DNS method using glucose as standard (table 30).

In this experiment used the following substrates:

KRISTALLIChESKAYa (Avicel);

The washed pretreated ferry fiber spruce (integrirovannoe 3%/SO2within 20 min, then pretreatment with steam at 215aboutC for 5 min), dry material 25,9% (SPRUCE);

The washed crude oxidized fiber, corn straw (WOCS);

The washed pretreated ferry fiber willow (pretreatment for 14 min at 210aboutC), dry matter 23,0% (WILLOW).

Table 30
The products of hydrolysis using CBH enzymes (45aboutC, pH 5.0). The reaction products after 48 h of hydrolysis in the form of reducing sugars (mg/ml), measured using glucose as standard. Abbreviations: CBH = cellobiohydrolase; EGI = endoglucanases I (Cel7B) of T. reesei, EGII = endoglucanases II (Cel5A) of T. reesei; bG = β-glucosidase (Novozym 188); XYL = xylanase pI 9 (XYN II) T. reesei; AXE = acetylcholin the esterase T. reesei; nd = not used
EnzymesSubstrates
CBHAdditional enzymesAvicelSPRUCEWOCSWILLOW
Ta Cel7AEGII, bG 2,02,02,82,0
Ta Cel7A+Tr CBDEGII, bG5,84,04,44,0
Ta Cel7A+Ct CBDEGII, bGa 4.93,74,63,7
At Cel7AEGII, bG5,33,34,53,3
Ct Cel7AEGII, bG6,02,63,42,6
Cel7A of T. reeseiEGII, bGthe 4.72,92,92,9
Ta Cel7AEGII, EGI, XYL, AXE, bGndnd4,32,8
Ta Cel7A+Tr CBDEGII, EGI, XYL, AXE, bGndnd 7,25,9
Ta Cel7A+Ct CBDEGII, EGI, XYL, AXE, bGndnd7,25,6
At Cel7AEGII, EGI, XYL, AXE, bGndnd6,4of 5.4
Ct Cel7AEGII, EGI, XYL, AXE, bGndnd5,64,0
Cel7A of T. reeseiEGII, EGI, XYL, AXE, bGndnd6,04,1

In Table 30 different cellobiohydrolase compared based on the same dose of protein hydrolysis. The results show that on cellulosic substrates (Avicel and fiber spruce) Cel7A Thermoascus aurantiacus with genetically attached CBD showed clearly higher hydrolysis than T. reesei CBHI/Cel7A. Without CBD T. aurantiacus Cel7A was less effective on these substrates. The effect of cellobiohydrolase Acremonium thermophilum and Chaetomium thermophilum was also better than the effect of T. reesei CBHI/Cel7A on multiple substrates, in particular C. thermophilum Cel7A high efficiency pure cellulose (Avicel).

In the case of substrates containing significant amounts of hemicelluloses (willow and corn straw), CBH/Cel7 enzyme clearly needed additionally in hemicellulose and endoglucanase to act effectively. If additional hemicellulase was not present, Cel7A of T. aurantiacus with genetically attached CBD again showed the highest hydrolysis. The most important hemicellu-degrading enzymes (xylanase, acetylcholin the esterase and EGI) Cel7A of T. aurantiacus with genetically attached CBD again operated with the highest efficiency. A. thermophilum Cel7A was more effective than the enzyme from T. reesei and C. thermophilum produced hydrolytic products at the same level as T. reesei CBHI/Cel7A. Pulp-binding domain of T. reesei, apparently, gives slightly better performance in the hydrolytic action of the T. aurantiacus Cel7A than CBD C. thermophilum, although the difference was quite small.

It can be concluded that when CBHI/Cel7A was replaced by a mixture of enzymes produced by Trichoderma here cellobiohydrolase, hydrolytic efficiency, based on experimental data, was clearly superior in the case of T. aurantiacus Cel7A with Genichesk attached CBD, and also improved in the case of A. thermophilum Cel7A and C. thermophilum Cel7A. If we take into account also the best temperature stability produced here cellobiohydrolase, the results indicate that the effect of mixtures of CE is lulashnyk enzymes at temperatures higher than 45aboutWith, can clearly be improved by the use made here of cellobiohydrolase.

Example 25. Effect of recombinant endoglucanases hydrolysis

Drugs, containing endoglucanase, compared to hydrolysis studies mixed with purified enzymes CBH/Cel7 and CBH/Cel6 on several pre-treated substrates. The filtrates of cultures of T. reesei containing various clones endoglucanase enzymes, obtained as described in Example 19. The enzymes were enriched by removing thermolabile proteins from mixtures by temperature treatment (60aboutS, 2 h, pH 5), and the supernatant was used for hydrolysis studies. In addition, enzyme mixtures used pure cellulase of T. reesei (purified as described Suurnäkki et al., 2000). In the experiment used the following endoglucanase:

The endoglucanases Acremonium thermophilum ALKO4245 At EG_40/Cel45A (ALKO4245 EG_40);

The endoglucanases Acremonium thermophilum ALKO4245 At EG_40_/Cel45B (ALKO4245 EG_40_);

The endoglucanases Thermoascus aurantiacus ALKO4242 Ta EG_28/Cel5A (ALKO4242 EG_28).

In this experiment used the following substrates:

The washed pretreated ferry fiber spruce (integrirovannoe 3% SO2within 20 min, then pretreatment with steam at 215aboutC for 5 min), dry material 25,9% (SPRUCE);

Torn ferry fiber, corn straw (SECS);

Endo is glucanase, be tested (at a dose of 840 ncat/g dry matter, based on endoglucanase activity against HEC in accordance with IUPAC, 1987), used either cellobiohydrolase T. reesei (CBHI/Cel7A, 8,1 mg/g dry. prophetic. and CBHII/Cel6A, 2.0 mg/g dry. prophetic.), or Thermoascus aurantiacus Cel7A with genetically attached CBD of T. reesei (10,1 mg/g dry. prophetic.). For comparison, experiments were also included purified (Suurnäkki et al., 2000) EGI (Cel7B) and EGII (Cel5A) of T. reesei. All mixtures were supplemented with β-glucosidase Novozym 188 (to make a total dose of β-glucosidase 500 ncat/g dry. weight.; relatively high dose used to compensate for the difference in background various activities EG drugs). In triplicate tubes were incubated while stirring at 45aboutC for 48 h and prepared control samples with inactivated enzymes and the corresponding substrates. The release of hydrolysis products was measured as reducing sugars by DNS method using glucose as standard (table 31).

Table 31
The products of hydrolysis with different endoglucanase drugs, used in conjunction with cellobiohydrolase from T. reesei or T. aurantiacus Cel7A containing CBD of T. reesei. The reaction products after 48 h of hydrolysis (45aboutC, pH 5.0) as reducing sugars (mg/ml), measured using glucose as standard. Abbreviations: CBHI = cellobiohydrolase (Cel7A) of T. reesei; CBHII = cellobiohydrolase (Cel6A) T. reesei; EGI = endoglucanases I (Cel7B) of T. reesei, EGII = endoglucanases II (Cel5A) of T. reesei; bG = β-glucosidase (Novozym 188); nd = not used
EnzymesSubstrates
The endoglucanasesCBH/Cel7SPRUCESECS
Without addingEG CBHI and CBHII of T. reesei2,43,2
EGICBHI and CBHII of T. reesei3,54,6
EGIICBHI and CBHII of T. reeseithe 3.83,5
At EG_40CBHI and CBHII of T. reeseia 4.94,3
At EG_40_CBHI and CBHII of T. reesei4,54,8
Ta EG_28CBHI and CBHII of T. reesei3,0a 3.9
Without adding EGT. aurantiacus Cel7A+Tr CBD1,82,1
EGIT. aurantiacus Cel7A+Tr CBDnd.4,2
EGIIT. aurantiacus Cel7A+Tr CBD3,2nd.
At EG_40T. aurantiacus Cel7A+Tr CBD4,84,0
Ta EG_28T. aurantiacus Cel7A+Tr CBD1,5nd.

In Table 31 different endoglucanase compared based on the same dose of activity in the hydrolysis. Perhaps this excessive action of enzymes with low specific activity against the substrate (hydroxyethyl cellulose)used in the analysis, and underestimated the effectiveness of enzymes with high specific activity against hydroxyethyl cellulose. In any case, the results show that endoglucanase Acremonium thermophilum work very well in the hydrolysis, when acting together with both cellobiohydrolase used in the mixture. Endoglucanase A. thermophilum have an action similar to the action of T. reesei EGI/Cel7B, which is very effective fer entom on hemicellulose-containing substrate from corn straw due to his strong side xylanase activity. The endoglucanases Cel5A T. aurantiacus (ALKO4242 EG_28) showed lower hydrolysis than the enzymes of T. reesei.

We can conclude that endoglucanase from A. thermophilum are comparable or improved performance compared with the corresponding Trichoderma enzymes in the hydrolysis, judging by this experiment. If we take into account also thermal stability of the produced here endoglucanases, the results indicate that the effect of mixtures of cellulase enzymes at temperatures higher than 45aboutWith, you can improve when you use described here endoglucanases.

Example 26. Hydrolysis of pretreated by steam fibers spruce at high temperatures

Washed expanded ferry fiber spruce (integrirovannoe 3%/SO2within 20 min, then pretreatment with steam at 215aboutC for 5 min) with a content of dry material of 25.9% suspended in 5 ml of 0.05 M sodium acetate buffer at a concentration of 10 mg/ml of This substrate is hydrolyzed using various enzyme mixtures in test tubes while stirring with a magnetic stirrer in a water bath with controlled temperature for 72 hours For each sample test tubes in triplicate were withdrawn from hydrolysis, boiled for 10 min to complete enzymatic hydrolysis, centrifuged and the supernatant analyzed n is the reaction products of hydrolysis. Blank samples containing only the substrate (only buffer was added instead of enzyme)was also incubated under the appropriate conditions.

A mixture of thermophilic cellulases prepared using the following components:

Thermophilic drug CBH/Cel7 containing Thermoascus aurantiacus ALKO4242 Cel7A c genetically attached CBD of T. reesei CBHI/Cel7A. Protein are unfamiliar with the preparation produced as described in Example 15, and was purified in accordance with Example 2, obtaining the purified product Ta Cel7A+Tr CBD with a protein content of 5.6 mg/ml;

The drug thermophilic endoglucanase containing endoglucanase At EG_40 Acremonium thermophilum 4245. The protein was obtained by T. reesei, as described in Example 19. To enrich thermophilic components, environment waste culture was treated by heating (60aboutC for 2 hours). The resulting preparation contained 4,9 mg/ml protein, and its endoglucanase activity was (according to IUPAC, 1987) 422 ncat/ml;

The drug thermophilic β-glucosidase, prepared as described in Example 21 containing β-glucosidase Ta βG_81/Cel3A Thermoascus aurantiacus ALKO4242. To enrich thermophilic components, fermentative broth was treated by heating (65aboutC for 2 hours). The resulting preparation contained 4.3 mg/ml protein, and its endoglucanase activity was (according to Bailey and Linko, 1990) 6270 ncat/ml

These enzyme preparations were combined footprint is accordingly (at 10 ml mixture): CBH/Cel7-drug 4,51 ml, endoglucanases drug 5,19 ml and β-glucosidases the preparation of 0.29 ml of This mixture was used as a "MIXTURE 1" thermophilic enzymes.

For comparison and control was made known from the prior art mixture of commercial enzymes Trichoderma reesei, includes (10 ml): 8,05 ml Celluclast 1.5 L FG (Novozymes A/S). She was labelled as T. REESEI ENZYMES".

The enzymes were dosed out on the basis of the FPU activity of mixtures: MIXTURE 1, using a dosage of 5.5 FPU per 1 gram of dry matter in the substrate fibers ate, and T. REESEI ENZYMES, using 5,8 FPU per 1 gram of dry matter in the substrate fibers ate.

The samples were taken from hydrolysis after 24, 48 and 72 h and treated as described above. The products of hydrolysis was quantified using the analysis of reducing sugars (Bernfeld, 1955), using glucose as standard. The amount of hydrolysis products, such as reducing sugars, presented in Figure 9.

The results clearly show the best action of the enzymes described here in comparison with the known from the prior art enzymes Trichoderma at 55 and 60aboutWith the spruce substrate. On the basis of HPCL analysis the maximum yield of sugars from the substrate would be 5,67 mg to 10 mg of dry spruce substrate. Due to the relatively low dosage of the enzyme, the final output of sugars was clearly reduced. For thermostable enzymes output of sugars on the basis of the analysis of reducing sugars was 66 and 57% of theoretical at 55 and 60 aboutC, respectively. For well-known from the prior art enzymes Trichoderma he was only 31 and 11% at 55 and 60aboutC, respectively.

Example 27. Hydrolysis of pretreated by steam fibers corn straw at high temperatures

Torn ferry fiber, corn straw (treatment at 195aboutC for 5 min) with a dry matter content of 45.3% suspended in 5 ml of 0.05 M sodium acetate buffer at a concentration of 10 mg/ml Processing and measurements were made as described in Example 26.

The mixture described here thermophilic cellulases prepared using the following components:

Thermophilic drug CBH containing Thermoascus aurantiacus ALKO4242 Cel7A c genetically attached CBD of T. reesei (CBHI/Cel7A Ta Cel7A+Tr CBD, Example 15). Protein content in the product was 31 mg/ml;

The drug thermophilic endoglucanase containing endoglucanase At EG_40/Cel45A Acremonium thermophilum 4245, obtained as described in Example 19. The concentrated enzyme preparation was endoglucanase activity (according to IUPAC, 1987) 2057 ncat/ml;

The drug thermophilic β-glucosidase containing β-glucosidase Ta βG_81/Cel3A Thermoascus aurantiacus ALKO4242, obtained as described in Example 21, he had β-glucosidase activity (according to Bailey and Linko, 1990) 11500 ncat/ml;

Thermophilic xylanases product containing the xylanase AM24 originating from Novomuraea flexuosa DSM43186. This PR is the product made, using recombinant strain of Trichoderma reesei, which was transformed expression cassette pALK1502, as described in WO 2005/100557. The solid product was dissolved in water to make 10%solution, and received an enzyme with xylanase activity (reviewed by Bailey et al., 1992) 208000 ncat/ml

These enzyme preparations were combined in the following way (10 ml mixture): CBH/Cel7-drug 7,79 ml, endoglucanases the preparation of 0.96 ml, and β-glucosidases drug 1,14 ml. of This mixture was used as a "MIXTURE 2" thermophilic enzymes.

For comparison and control was made known from the prior art mixture of commercial enzymes Trichoderma reesei, includes (10 ml): 8,05 ml Celluclast 1.5 L FG (Novozymes A/S) and 1.95 ml of Novozyme 188 (Novozymes A/S). She was labelled as T. REESEI ENZYMES".

The samples were taken from hydrolysis after 24, 48 and 72 h and treated as described above. The products of hydrolysis was quantified using the analysis of reducing sugars (Bernfeld, 1955), using glucose as standard. The results from the blank substrate was subtracted from the sample with the enzyme, and the concentration of the hydrolysis products, such as reducing sugars, presented in Figure 10.

The results clearly show the best action of the enzymes described here in comparison with the known from the prior art enzymes Trichoderma. At 45aboutWith a mixture of thermophilic enzymes show is and the more efficient hydrolysis compared with enzymes T. reesei: the hydrolysis was faster and, in addition, was obtained higher yield of sugars. Based on HPLC analysis the maximum yield of sugars (including available soluble sugar naramata the substrate that was used) of the substrate would be 5,73 mg to 10 mg of dry substrate. Thus, hydrolysis using an enzyme MIXTURE 2 was almost complete within 48 hours. At 55 and 60aboutDescribed here thermophilic enzymes also showed clearly the best action in the hydrolysis compared with the known from the prior art enzymes Trichoderma.

Example 28. Hydrolysis of pre-treated at high temperature fiber, corn straw using a mixture of thermostable xylanase

Repeating the procedure described in Example 27, except that xylanases product XT 02026A3 was replaced by the preparation of thermostable xylanase containing the xylanase Ta XYN_30/Xyn10A Thermoascus aurantiacus ALKO4242 produced in T. reesei. Enzyme broth, obtained as described in Example 23, had xylanase activity 132000 ncat/ml were analyzed by Bailey et al., 1992).

These enzyme preparations were combined in the following way (10 ml mixture): CBH/Cel7-preparation of 7.64 ml, endoglucanases the preparation of 0.96 ml, β-glucosidases the preparation of 1.15 ml and xylanases the drug of 0.25 ml of This mixture was used as a "MIX 3" thermophilic enzymes.

For sravnenie and control was made known from the prior art mixture of commercial enzymes Trichoderma reesei, includes (10 ml): 8,05 ml Celluclast 1.5 L FG (Novozymes A/S) and 1.95 ml of Novozyme 188 (Novozymes A/S). She was labelled as T. REESEI ENZYMES".

The samples were taken from hydrolysis after 24, 48 and 72 h and treated as described above. The products of hydrolysis was quantified using the analysis of reducing sugars (Bernfeld, 1955), using glucose as standard. The results from the blank substrate was subtracted from the sample with the enzyme, and the concentration of the hydrolysis products, such as reducing sugars, presented in Figure 11.

The results clearly show the best action of the enzymes described here in comparison with the known from the prior art enzymes Trichoderma. At 45aboutWith a mixture of thermophilic enzymes showed more efficient hydrolysis compared with enzymes of T. reesei. At 55 and 60aboutDescribed here thermophilic enzymes also showed clearly the best action in the hydrolysis compared with the known from the prior art enzymes Trichoderma. Action new Fermentas mixture at 60aboutWith was on the same level as the known from the prior art enzymes at 45aboutC.

Example 29. Hydrolysis of pre-treated at high temperatures of spruce fibers using a mixture of thermostable xylanase

The procedure as described in Example 28 was repeated with washed vspuchennym ferry fiber spruce (impregned the bath 3%/SO 2within 20 min, then pretreatment with steam at 215aboutC for 5 min, with a dry matter content of 25.9%) as substrate, using temperature hydrolysis 45, 55 and 60aboutC. Samples were removed from the hydrolysis after 24, 48 and 72 h and processed as described above. The products of hydrolysis was quantified using the analysis of reducing sugars (Bernfeld, 1955), using glucose as standard. The results from the blank substrate was subtracted from the sample with the enzyme, and the concentration of the hydrolysis products, such as reducing sugars, presented in Figure 12.

The results clearly show the best action of the mixture described herein enzymes in comparison with the known from the prior art enzymes Trichoderma at the investigated temperatures. At 45aboutWith a mixture of thermophilic enzymes showed more efficient hydrolysis compared with enzymes of T. reesei, obviously, due to the higher stability during long time of hydrolysis. When 55aboutWith the effectiveness of the mixture described herein enzymes was still on the same level as at 45aboutSince, as known from the prior art mixture was inefficient with the substrate used at this temperature. At 60aboutDescribed here thermophilic enzymes showed a reduced hydrolysis, although the hydrolysis was almost on the same level as the effects of the world is different from the prior art enzymes at 45 aboutC.

Example 30. Evaluation of glucose inhibition of β-glucosidase from Acremonium thermophilum, ALKO4245, Chaetomium thermophilum ALKO4261 and Thermoascus aurantiacus ALKO4242

The filtrates of the cultures produced by strains of Acremonium thermophilum, ALKO4245, Chaetomium thermophilum ALKO4261 and Thermoascus aurantiacus ALKO4242 described in Example 1. β-glucosidase activity (measured in accordance with Bailey and Linko, 1990) of these drugs was 21.4, 5,6 and 18.6 ncat/ml, respectively. For comparison in the experiment were also included commercial enzymes Celluclast 1.5 L (β-glucosidase 534 ncat/ml) and Novozyme 188 (β-glucosidase 5840 ncat/ml).

In order to assess the sensitivity of various β-glucosidase to glucose inhibition, the standard procedure of analysis of the activity performed in the presence of different concentrations of glucose. Substrate solution (p-nitrophenyl-β-D-glucopyranosid) for analysis of β-glucosidase activity was supplemented with glucose so that the glucose concentration in the analytical mixture was adjusted to values of from 0 to 0.5 M except that adding glucose analysis was performed using standard procedure (Bailey and Linko, 1990). Activity in the presence of varying concentrations of glucose as a percentage of the activity without glucose are presented in Figure 13.

The results show that β-glucosidase from C. thermophilum and T. aurantiacus were less susceptible to inhibition by glucose than β-glucosidase, presence is adequate in the commercial enzymes: originating from Aspergillus β-glucosidase in Novozyme 188 or originating from Trichoderma β-glucosidase in Celluclast 1,5 L. The enzyme from A. thermophilum showed a behavior comparable to the enzyme of T. reesei Celluclast. Especially the enzyme of C. thermophilum was clearly less effective at high concentration of glucose. Thus, these results suggest that accounting glucose inhibition when using the new β-glucosidase, especially from strains of Acremonium thermophilum ALKO4242 and Chaetomium thermophilum ALKO4261, would give a clear advantage in the hydrolysis in an industrial environment with a high concentration of glucose.

Example 31. Activity of filter paper in enzyme mixtures at high temperatures

Activity Ferroalloy paper in the enzyme preparations was measured according to IUPAC (1987), as described in the Protocol except that the enzymatic reaction was carried out at temperatures from 50 to 70aboutC. Calculated FPU activity is based on the amount of enzyme required to hydrolyse 4% of the substrate of the filter paper for 1 h in the experiment. It is believed that the FPU activity is the total activity of all cellulases enzyme preparation.

Used enzyme mixture: a MIXTURE of 2, prepared as described in Example 27, a MIXTURE of 3, prepared as described in Example 28, and the MIXTURE 4. A MIXTURE of 4 prepared by combining enzyme preparations described in Example 27, the following (10 ml mixture): CBH/Cel7-drug to 7.84 ml, endoglucanases drug 0,99 ml of the β-glucosidase drug 1,17 ml.

As control was used the following enzyme mixture, representing a mixture of known from the prior art:

"T. REESEI ENZYMES AND cooked as a drug "T. REESEI ENZYMES as described in Example 26;

"T. REESEI ENZYMES IN the"drawn up by the combination (10 ml) 8,05 Econase CE (commercial cellulase preparation T. reesei from AB Enzymes Oy, Rajamäki, Finland) and 1.95 ml of Novozyme 188 (Novozymes A/S).

FPU activity of enzyme preparations, measured at different temperatures, are presented in Figure 14 as a percentage of the activity in the standard (IUPAC, 1987) conditions (50aboutC).

The results clearly show that the mixtures according to the invention find a higher total cellulase activity at high (60-70aboutC) temperatures in comparison with the known from the prior art mixtures, based on the enzymes from Trichoderma or Aspergillus.

Example 32. The application of new beta-glucosidase for cooking sophorose

A mixture with a high concentration of starch hydrolysate (Nutriose 74/968, Roquette) was treated βG_81/Cel3A Thermoascus aurantiacus, enriched enzyme preparation produced as described in Example 21 to obtain a mixture of sugars containing significant amounts of cellulase inducer (sophorose) to overcome glucose repression.

Enriched Ta βG_81/Cel3A enzyme preparation was added to a 70%increase (in/in) to a solution Nutriose to the end to which ncentratio 1 g total protein per liter. The container with the mixture incubated in a water bath at 65aboutC for 3 days with constant stirring and was used as carbon source in the medium shake flasks for two different Trichoderma-strains (A47 and Rut-C30). Effect of enzyme treatment was measured as endoglucanase activity, formed within 7 days of cultivation in shake flask. As a control the cultivation was performed under the same conditions with raw Nutrioso as a carbon source. Under cultivation in shake flasks, put on Ta βG_81/Cel3A pre-treated with tested strains Nutrional environment, received a more than twofold increase in activity. The results are shown in Figure 15.

A list of the deposited organisms

CBS(1)
StrainContained a plasmidDepositary authorityDate DepositDepository number
Acremonium thermophilum ALKO4245-CBS(1)20 Sep. 2004CBS 116240
Thermoascus aurantiacus ALKO4242-20 Sep. 2004CBS 116239
Chaetomium thermophilum ALKO4265-CBS(2)8 Nov. 1995CBS 730.95(4)
Escherichia colipALK1635DSMZ(3)16 Sep. 2004DSM 16723
Escherichia colipALK1642DSMZ16 Sep. 2004DSM 16727
Escherichia colipALK1646DSMZ16 Sep. 2004DSM 16728
Escherichia colipALK1861DSMZ16 Sep. 2004DSM 16729
Escherichia colipALK1715DSMZ16 Sep. 2004DSM 16724
Escherichia colipALK1723DSMZ16 Sep. 2004DSM 16725
Eschericia coli pALK1725DSMZ16 Sep. 2004DSM 16726
Escherichia colipALK1904DSMZmay 13, 2005DSM 17323
Escherichia colipALK1908DSMZmay 13, 2005DSM 17324
Escherichia colipALK1925DSMZmay 13, 2005DSM 17325
Escherichia colipALK1926DSMZmay 13, 2005DSM 17326
Escherichia colipALK2001DSMZ18 Oct. 2005DSM 17667
Escherichia colipALK2010DSMZ18 Nov. 2005DSM 17729
(1)The Central Bureau of Cell Cultures in Uppsalalaaan 8, 3584 CT, Utrecht, the Netherlands;
(2)The Central Bureau of Cell Cultures in Oosterstraat 1, 3742 SK BAARN, Nida is Landes;
(3)German Collection of Microorganisms and Cell cultures GmbH (DSMZ), Mascheroder Weg 1 b, D-38124 Braunschweig, Germany;
(4)[At the end of the current period of storage, the samples will be stored under the agreement, making the strain available after expiration of the patent].

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1. The polypeptide of cellobiohydrolase selected from the group consisting of:
a) a polypeptide comprising amino acid sequence having at least 95%identity to SEQ ID NO:6 and having cellobiohydrolase what aktivnosti, and
b) a fragment of (a)having cellobiohydrolase activity.

2. The polypeptide according to claim 1 including:
a) amino acid sequence having at least 99%identity to SEQ ID NO:6; or
b) a fragment of (a)having cellobiohydrolase activity.

3. Selected polynucleotide encoding cellobiohydrolase selected from the group consisting of:
a) the nucleotide sequence of SEQ ID NO:5 and sequences that encodes a polypeptide according to claim 1.

4. The expression vector encoding cellobiohydrolase according to claim 1 and including polynucleotide according to claim 3.

5. A host cell belonging to filamentous fungi, containing the vector according to claim 4 and expressing cellobiohydrolase.

6. A host cell according to claim 5, which is able to Express the polypeptide according to claim 1 encoded by a heterologous polynucleotide sequence according to claim 3.

7. A host cell according to claim 6, which represents the cell strain of the genus Trichoderma or Aspergillus.

8. The Escherichia coli strain comprising a heterologous polynucleotide containing the gene that encodes the protein according to claim 1, and having access number DSM 16729.

9. The enzyme preparation having cellulolytic activity, comprising a polypeptide according to claim 1 and optionally other enzyme polypeptides having cellobiohydrolase, endoglucanase, beta-glucosidase or cialisnow activity, with specified shall report received by way comprising culturing the host cell according to claim 5, which was transformed expressing vector coding for the specified polypeptide under conditions ensuring the receipt of the said polypeptide and recovering the culture medium containing the polypeptide, and optionally highlight of her host cell.

10. The enzyme preparation according to claim 9, which is represented in the form of the spent medium of cultivation, powder, granules or liquid.

11. The enzyme preparation according to claim 9, which additionally contains conventional additives selected from the group including mediators, stabilizers, preservatives, sufactant, buffers, and parts of the environment of cultivation.

12. The enzyme preparation according to claim 9, further comprising endoglucanase, beta-glucosidase, and xylanase.

13. The enzyme preparation according to item 12, where the endoglucanases includes an amino acid sequence having at least 95%identity to SEQ ID NO:10, 12, 14 or 16 and having endoglucanase activity, or its enzymatically active fragment.

14. The enzyme preparation according to item 12, where beta-glucosidase comprises amino acid sequence having at least 95%identity to SEQ ID NO:22, 24 or 26 and with beta-glucosidase activity, or its enzymatically active fragment.

15. The enzyme preparation according to item 12, where xylanase includes aminokislotnoi sequence, having at least 95%identity to SEQ ID NO:18 or 20 having a xylanase activity, or its enzymatically active fragment.

16. The use of the polypeptide according to claim 1 or enzyme preparation according to claim 9 for the degradation of cellulosic material.

17. The application of article 16, where the cellulosic material is selected from the group consisting of corn straw, millet, straw, grains, husks sugarcane and originating from wood material.

18. The application of article 16 in the fuel, pulp and paper industry or the production of fodder.

19. Use p, where the enzyme is used in the treatment of Kraft pulp, mechanical pulp or recycled paper.

20. Use p, where an enzyme is an old cultural medium.

21. The method of producing the polypeptide according to claim 1, comprising transforming a host cell according to claim 5 vector coding for the said polypeptide, and culturing this host cell under conditions enabling expression of the above-mentioned polypeptide, and optionally the extraction and purification of the produced polypeptide.

22. The method of processing cellulosic waste material environment culturing the host cell according to claim 5, capable of producing the polypeptide according to claim 1, which comprises reacting the cellulosic material with well-established among the first cultivation to obtain a hydrolyzed cellulose material.

23. The method of processing cellulosic material with an enzyme preparation according to claim 9, containing the polypeptide of cellobiohydrolase according to claim 1 together with endoglucanases and beta glucanase.

24. The method according to item 23, in which cellobiohydrolase obtained from Acremonium thermophilum.

25. The method according to paragraph 24, in which cellobiohydrolase obtained from a strain of Acremonium thermophilum CBS 116240.

26. The method according to item 23, in which the endoglucanases includes an amino acid sequence having at least 95%identity to SEQ ID NO: 10, 12, 14 or 16 and having endoglucanase activity, or its enzymatically active fragment.

27. The method according to item 23, in which the beta-glucosidase comprises amino acid sequence having at least 95%identity to SEQ ID NO:22, 24 or 26 and having beta-glucosidase activity, or its enzymatically active fragment.

28. The method according to item 23, in which the cellulosic material is a lignocellulosic material.

29. The method according to p, including the processing of lignocellulosic material with at least one additional enzyme, preferably xylanase, which preferably includes an amino acid sequence having at least 95%identity to SEQ ID NO:18 or 20 and having xylanase activity or its enzymatically active fragment.

30. The method according to item 23, in which the enzymes to ablaut to cellulosic material, or both, or sequentially.

31. The method according to item 23, in which the cellulosic material is selected from the group consisting of corn straw, millet, straw, grains, husks sugarcane and originating from wood material.

32. Application of the method according to item 23 in the process of producing ethanol from cellulosic material.



 

Same patents:

FIELD: food industry.

SUBSTANCE: vapour from the boiler is supplied into the bottom part of distillation column through the bubble flask. Wine material from the gravity vessel is supplied into the dephlegmator through a rotameter; the dephlegmator is represented by a heat exchanger with horizontal tubes wherethrough the wine material passes; alcohol-water vapours rising up through the distillation column are delivered into the intertube space; due to the vapours condensation the wine material heating is performed. The wine material heated up to 75-80°C is supplied, through the distribution device, into the distillation column and onto the nozzle ring with porcelain contact devices; during flowing down onto the nozzle ring with single-cap double boiling plates alcohol is completely removed from the material. Generated alcohol-water vapours rise up through the distillation column to the ring with multi-cap plates and consolidate due to interaction with phlegma flowing down through the distillation column. The alcohol-water vapours are dephlegmated and condensed in the dephlegmator and condenser. A part of condensate generated in the condenser in the form of heads draw is cooled in the refrigerator and supplied into the drain section. The other part of condensate is returned in the form of phlegma into the distillation column and onto the upper plate of the ring with multi-cap plates, alcohol-water vapours are sprayed and consolidated with this condensate. Condensate generated in the dephlegmator is cooled in the refrigerator to produce cognac alcohol with an alcoholic content of 62-70 vol. %. Slop generated in the distillation process is discharged from the distillation column blocking vapour outlet from it. The installation performance is 900-1000 dhal of absolute alcohol per day, vapour rate is 12-15 kg/dhal, pressure in the column bottom is 70-80 kPa. The produced cognac alcohol indices are the follows: colour - colourless; aroma and taste - without foreign smells and after-tastes; weight concentration of higher alcohols - 420 mg/100 cm3, medium esters - 90 mg/100 cm3, volatile acids - 40 mg/100 cm3.

EFFECT: invention ensures the process continuality, accelerates and simplifies the process due to the installation performance enhancement.

2 cl, 2 ex

FIELD: food industry.

SUBSTANCE: potatoes are thermally treated at a temperature of 0°C and below during from 20 days and longer. The potatoes are milled into mush and undergo fermentative saccharification. Optionally, potatoes thermal treatment at a temperature of 0°C and below may be combined with potatoes storage at a raw material storage facility.

EFFECT: invention ensures minimal energy expenditure, exclusion of sugars thermal destruction and melanoidin -generating reaction.

2 cl, 6 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a Saccharomyces cerevisiae VKPM Y-3415 yeas strain which produces ethyl alcohol. The strain is adapted to culturing on dairy cheese whey.

EFFECT: strain is capable of producing ethyl alcohol with high output and is antagonistic to accompanying microflora.

1 ex

FIELD: food industry.

SUBSTANCE: ethyl alcohol production method envisages peeling grains to produce a peel fraction in an amount of 2-5% wt of the total weight of grains and a fraction containing endosperm, milling the fraction containing endosperm, mixing the milled fraction containing endosperm with water, its three-stage water-and-heat treatment and enzymic hydrolysis, fermentation, fermented brew distillation to produce ethyl alcohol; milling is performed according to a closed circuit scheme envisaging passing the fraction containing endosperm through a hammer crusher with subsequent straining through a 1 mm diameter cell sieve to produce the through-product and the overtail; then the through-product is directed for mixing with water while the overtail is mixed with a new portion of the fraction containing endosperm and undergoes recrushing; the process of milling and purifying is repeatedly performed till formation of 100% through-product through of the total of the initial fraction containing endosperm a 1 mm diameter cell sieve.

EFFECT: invention allows to simplify the method for production of ethyl alcohol combined with preservation of its high quality characteristics and intensify the fermentation process.

3 ex

FIELD: food industry.

SUBSTANCE: ethyl alcohol production method envisages peeling grains to produce a peel fraction in an amount of 2-5% wt of the total weight of grains and a fraction containing endosperm, mixing the fraction containing endosperm with water at hydraulic module equal to 1:(3.5-4.0), introduction of amylolytic enzymic preparations of diluent and saccharifying action in an amount of 0.5-1.0 "АС" units and 2.0-3.0 "ГлС" units per 1 g of conventional starch, the mixture dispersion in a rotary-pulsation apparatus at treatment rate equal to 1.5-3.0*103 rpm during 5-10 minutes at a temperature of 56-58°C to produce the batch, the batch treatment at 56-58°C during 2-2.5 hours to produce saccharified wort, its cooling, brew fermentation and distillation to produce ethyl alcohol.

EFFECT: invention allows to reduce enzymic preparations consumption and decrease duration of water-and-heat and enzyme treatment.

1 tbl, 3 ex

FIELD: biochemistry.

SUBSTANCE: the yeast mixture used for high concentrated alcohol fermentation from sugar-containing material contains any kind of dry yeast and nutrients necessary for yeast growth. The nutrients includes 40-70 weight parts of dry yeast, 20-40 weight parts of nitrogen source, 5-10 weight parts of phosphorus source, 2.5-5 weight parts of inorganic salt different form the phosphorus-containing and nitrogen-containing inorganic salts, 1-2.5 weight parts of microvitamin and 0.5-1.2 weight parts of penicillin. The yeast is taken from the yeast group containing beer yeast Saccharomyces cerevisiae Hansen from the Saccharomyces cerevisiae species, vine yeast Saccharomyces uvarum Beijerinek. The yeast mixture is received through grinding the nutrients separately, excluding the dry yeast. The dry yeast is mixed with the grinded nutrients with a certain weight ratio, then the mixture is stirred mechanically or manually until it is homogeneous.

EFFECT: increased alcohol fermentation up to 14.5-15.7% in standard raw material, such as sucrose, and reduced sugar residue in fermented mesh down to 0 - 0.1%.

9 cl, 3 tbl, 6 ex

FIELD: food industry.

SUBSTANCE: installation includes a distillation column (1) and an externally heated reboiler (13) connected to the still (9) of the distillation column (1) and serving for supply of heat to the distillation column. The installation is equipped with a separator for separation of distillery slop into solid substance fraction and liquid fraction; the separator is designed, for example, in the form of a decanter. The reboiler (13) is designed in the form of an evaporating apparatus with a dropping film evaporating at least part of liquid juice fraction and supplying liquid juice fraction after evaporation and/or flash steam produced in the process of evaporation into the still (9) of the distillation column (1).

EFFECT: invention ensures supply of energy into the distillation column at a relative low temperature level without the risk of the reboiler contamination.

8 cl, 2 dwg

FIELD: food industry.

SUBSTANCE: method envisages grains purification from metal and adventitious impurities with their subsequent washing with cold water and separating impurities from the grains mass; the separated impurities differ from the grains mass in hydrodynamic properties. Then the grains are disinfected during 10-15 minutes at a temperature of 15-20°C in a vessel for ozonation by way of supplying ozonised water into it. The grains are soaked till moisture content is 37-39%, then one precedes with hydromilling and subsequent kneading using enzyme preparations of α-amylase and glucoamylase, then one precedes with water-and-heat and enzyme treatment of grains at a temperature of 60-62°C during approximately 3 hours while the produced wort is fermented at a temperature of 28-30°C during 60-66 hours.

EFFECT: method allows to reduce consumption of heat and cooling water.

4 cl, 2 dwg

FIELD: food industry.

SUBSTANCE: method envisages milling raw material, preparation of a premix, addition of an antiseptic agent, dilution and saccharification of the premix, wort fermentation and wash rectification. The raw material is milled to produce a fraction with particle size less than 1 mm while the operations of the premix preparation, its dilution, saccharification and wort fermentation are performed at a temperature no higher than 62.0°C. In the process of the premix preparation operation one introduces into the raw material α-amylase and up to 70% of cellulose, xylanase and beta-glucanase. The premix is saccharified with glucoamylase; at the closing stage of saccharification one introduces protease and the remaining portion of cellulose, xylanase and beta-glucanase.

EFFECT: invention allows to improve quality and organoleptic properties of alcohol due to imparting to it the taste and flavour of the initial grain raw material.

3 cl

FIELD: food industry.

SUBSTANCE: method provides for distillation of wash in the wash column, epuration of wash distillate with supply of hydraulic selection water onto the upper plate of the epuration column having a splitter, a digestion and a concentration parts, rectification of epurate in the rectification column with extraction of fusel oil fractions, fusel alcohol and non-pasteurised alcohol, cleaning rectified alcohol of methanol and head admixtures in the methanol column. Vapours of fusel oil fractions from the epuration and rectification columns are condensed in the fusel condenser. Vapour condensate from the fusel condenser is delaminated into two liquid phases in the decanter; it is from the lower water stratum that fusel oil fractions from the epuration column splitter part are delivered into the bleeding zone. The fraction from the epuration column condenser, part of vapour condensate from the said column reflux exchanger and fusel alcohol fraction are distilled in the distillate column working according to the hydraulic selection method and having a splitter, a digestion and a concentration parts. A fraction is bled from the distillate column, joined with the upper liquid stratum from the reflux exchanger and discharged from the wash-rectification system in the form of head and intermediate admixtures fraction. From the liquid phase of the lower plates of the distillate column digestion part ethanol fraction is discharged and directed onto the upper plate of the epuration column splitter part. Fractions from the wash, methanol and rectification columns condensers, the carbon dioxide separator condenser and the alcohol traps are distilled in the column for concentration of methanol and head impurities from the condenser whereof one bleeds head impurities concentrate discharged from the wash-rectification installation while still liquor is returned into the wash.

EFFECT: invention allows to enhance organoleptic properties of rectified alcohol and increased its yield.

1 dwg, 1 tbl, 2 ex

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

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.

EFFECT: invention provides producing high-active complex enzymatic preparations of glucoamylase and xylanase with high activity of xylanase with respect to an initial strain to be applied in various fields of agricultural sector.

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.

EFFECT: application of the claimed means enables to accelerate the process of cellulose substrate enzymatic cleavage, to increase the yield of target product and reduce expenditure of enzyme preparation.

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.

EFFECT: new bacteria strain is obtained, which is a producer of pectolytic Aspergillus foetidus 379- K (VKPM F-962) enzymes.

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.

SUBSTANCE: fermental preparation of a complex cellulase, xylanase and xyloglucanase for hydrolysis of cellulose and hemicellulose is obtained by propagation of the Penicillium verruculosum VKM F-3972D strain on the water medium containing in g/l: cellulose - 50-60, glucose - 10-30, mineral salts - KH2PO4 8-12, (NH2)2SO4 3-7, MgSO4·7H2O 0.2-0.4, CaCl2·2H2O 0.2-0.4, at continuous feeding with glucose. The propagation process is conducted at temperature of 26-30°C and pH in a range of 4.5-5.0. The culture liquid containing a fermental preparation, is separated in 120-144 h after propagation beginning, treated with ultrafiltration and dried up.

EFFECT: high specific activity of the complex of cellulase, xylanase and xyloglucanase and rising of efficiency of use of a fermental preparation in various areas of biotechnology.

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.

EFFECT: it is increased the output of acid protease and complex of carbohydrase.

2 ex

FIELD: biotechnology, biochemistry, enzymology.

SUBSTANCE: strain of mycelial fungus is prepared by the multistep mutagenesis and selection of the parent culture Penicillium funiculosum BKM F-3661. Prepared strain is cultured on liquid nutrient medium under conditions of continuous feeding with glucose. In 120 h after culturing cultural fluid is separated and concentrated by ultrafiltration and a liquid enzyme preparation or dried is obtained. Invention can be used for preparing fermented sugars designated for processing to ethanol, and as fodder supplements also.

EFFECT: valuable biological properties of fungus strain.

3 cl, 4 ex

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

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