Strain penicillium funiculosum producing enzyme complex such as cellulase, endo-1,4-beta-xylanase, cellobiohydrolase, beta- glucosidase, endo-1,3(4)-beta-glucanase, feruloylesterase

FIELD: biotechnology, in particular production of feed supplement and feed for farmer animals.

SUBSTANCE: Invention relates to strain Penicillium funiculosum producing enzyme complex such as cellulase, endo-1,4-beta-xylanase, cellobiohydrolase, beta-glucosidase, endo-1,3(4)-beta-glucanase, feruloyl-esterase. Liquid feed supplement for farmer animals such as poultry, pigs and ruminants contains microbial product obtained from strain Penicillium funiculosum and optionally enzymes disclosed in claim 1, antimicrobial agent, sorbitol, antifreeze, concentrated filtered fermentation broth in specific ratio. Dry feed for farmer animals such as poultry, pigs and ruminants contains microbial product obtained from strain Penicillium funiculosum and optionally enzymes disclosed in claim 1, carrier, components of dry fermentation broth in specific ratio.

EFFECT: improved accessibility of feed and amino acids; reduced phosphorus and ammonia emanation.

25 cl, 4 dwg, 32 tbl, 6 ex

 

The scope of the invention

The present invention relates to a new microorganism, new enzymes and new mixture of enzymes. In addition, the present invention relates to compositions of a mixture of enzymes, to its preparation and use in the food industry, food production and other industries, including (but not limited to, paper and textile industry.

Background of the invention

For a long time the enzymes used for a wide range of different industrial applications. Well-known examples of the baking industry, wine-making and the production of fruit juices (where enzymes are used for the destruction of pectins and β-glucans), textile industry (where cellulase is used for soft and smooth pulp tissue) and also that is not the last application, for preparation of animal feed. In this case, the enzymes improve digestion of animal feed from plant sources.

The last application provides a more efficient digestion of animal feed. The value of the feed can be determined by FCR (Feed Conversation Ratio = cost of feed per unit of output), the nutritional value, the quantity of feed in relation to the increase in weight of the animal. The decrease in FCR feed points on a Pro rata among the elicina weight of the animal, meaning that the animal is able to use food more efficiently.

Poor digestibility of feed components (starch, fat, protein/amino acids) is a distinctive feature of feed based on cereals, for example, especially those that have high content of barley or wheat. In these cases, it may be necessary to prepare food so that they contain higher levels of energy from other sources and other additives, such as amino acids. These enzymes increase the amount of apparent metabolizable energy (Apparent Metabolizabie Energy)included in the forage grass.

Another approach to solving this problem was the introduction of enzyme additives, cellulases, endo-1,3(4)-β-glucanase (β-glucanase, endo-1,4-β-xylanase (xylanase), etc. or mixtures of enzyme activities such feed based on cereals. Enzyme supplements can have a specific use for hydrolysis β-glucans or for hydrolysis of arabinoxylans located in grain (usually barley and wheat). The addition of enzymes has a different purpose. One of the advantages, which, of course, ensures the effectiveness of enzyme additives to fodder, is to reduce the viscosity of the materials in the digestive tract of animals that receive food based on cereals, containing the appropriate enzyme Supplement. Higher elm is ity due in part, the presence of β-glucans and arabinoxylans in barley and wheat. Lower viscosity resulting from the action of enzymes, allows for easier absorption of nutrients in the digestive tract of animals. Another advantage is the selection of nutrients, prisoners in the cell walls of cereals, which reduces the need for other expensive dietary supplements. The overall result is a significant reduction in feed cost with the same or greater effect on FCR measurement.

It was revealed that the enzyme preparations obtained from a wide variety of microorganisms, increase the digestibility of the feed.

Given the known level associated with the use of enzymes in animal feed, the authors of the European patent application No. 0699762, in which is disclosed the use of phytase isolated from Schwanniomyces occidentalis. This phytase is a phytase derived from genetically modified organism, obtained by integration of the cloned gene, which the authors would like to avoid in the present invention.

With regard to patent applications WO 95/26398, there's also a modified cellulase is obtained by integration of the foreign DNA sequence into the cell host, which modifies the nature of the initial strain, which is selected from the following list of organisms: Bacillus, Streptomyces, Saccharomyces, Schizosacch aromyces, Aspergillus. The main purpose of the authors of the present invention was to avoid the incorporation of a foreign gene in a microorganism which produces the enzyme.

In the patent application WO 96/05739 mixture of enzymes (xylanase, protease and do not necessarily β-glucanase) derived from different microorganisms. The authors give the example (page 5) a mixture of enzymes with respect Xylenol activity to β-glucanase activity of about 1:5. It was found that when xylase included in the diet of cereal-based optimal dose or so, the joint presence of enzymes, processorsa β-glucanase activity, improves FCR feed, which, of course, disadvantageous. Accordingly, the authors argue against the presence of β-glucanase, they recommend a maximum ratio of xylanase activity to β-glucanase activity 1:0-0,25.

In some cases, in order to ensure the presence of all the enzyme activities associated with the use of feed, it is necessary to obtain drugs from drugs derived from more than one microorganism. In some cases, the enzyme preparations were obtained from microorganisms subjected to genetic modification using recombinant DNA techniques.

The authors found a new microorganism belonging to the class of Penicillum funiculosum, which which contains new enzymes, and developed a mixture of enzyme activities, which can be successfully used to increase, mainly, the digestibility of animal feed based on cereals.

Summary of invention

Accordingly, the present invention relates to a new microorganism, obtained from Penicillum funiculosum, and to the method of cultivation of the microorganism and the selection of enzymes produced by this microorganism.

In addition, in accordance with the present invention proposed new enzymes isolated from this organism, the nucleic acid sequences and compositions containing these enzymes.

Further, in accordance with the present invention, a method for increasing the digestibility of amino acids and animal feed based on amino acids and cereals.

Another objective of the present invention is a decrease in the allocation of phosphorus and excretion of ammonia from the cell of the battery, where they feed animals.

Detailed description of the invention

A. a New strain of Penicillum funiculosum

This new strain of fungus Penicillum funiculosum deposited under registration number M in ismaster International Depositories in accordance with the Budapest Agreement (1977), the International Institute of Mycology (International Mycological Institute (IMI), Bakeham Lane, Englefield Green, Egham, Surrey, TW209TY, UK).

Origin

A new strain of p is obtained from Penicillum funiculosum IMI 134756 after a successful UV irradiation and β -irradiation dispute, including screening on selective medium. No genetic modification was not obtained using recombinant DNA techniques using the embed alien DNA or RNA.

Identification and typing

Penicillum funiculosum IMI 134756 was characterized by growing on Czapek Dox agar at 25°C. the Characteristics of the colony, and micromorphology typical Penicillum funiculosum. Identification of the microorganism as Penicillum funiculosum was confirmed at the International Institute of Mycology (International Mycological Institute (IMI), Bakeham Lane, Englefield Green, Egham, Surrey, TW209TY, UK). Growth similar to dense basal felt with the foliar growth in the form of threads or knots of hyphae (funiculose), mycelium white with matte red in the substrate, the fields on the contrary pale but red-colored towards the centers may become dark red. This is a typical penicillin, it shows a short conidiophores arising mainly from the funicles, biverticillate, needle comedigenic cells, conidia elliptical and smooth.

The microorganism used to produce the drug enzymes of the present invention are grown under aerobic conditions in an environment that contains pulp, syrup from soaking corn, calcium carbonate and ammonium sulfate.

C. the fermentation Process

screening on selective medium. No genetically the modifications were not obtained using recombinant DNA techniques using the embed alien DNA or RNA.

Identification and typing

Penicillum funiculosum IMI 378536 was characterized by growing on Czapek Dox agar at 25°C. the Characteristics of the colony, and micromorphology typical Penicillum funiculosum. Identification of the microorganism as Penicillum funiculosum was confirmed at the International Institute of Mycology (International Mycological Institute (IMI), Bakeham Lane, Engiefield Green, Egham, Surrey, TW209TY, UK). Growth similar to dense basal felt with the foliar growth, in the form of threads or knots of hyphae (funiculose), mycelium white with matte red in the substrate, the fields on the contrary pale but red-colored towards the centers may become dark red. This is a typical penicillin, it shows a short conidiophores arising mainly from the funicles, biverticillate, needle comedigenic cells, conidia elliptical and smooth.

The microorganism used to produce the drug enzymes of the present invention are grown under aerobic conditions in an environment that contains pulp, syrup from soaking corn, calcium carbonate and ammonium sulfate.

C. the fermentation Process

These new fungi is produced by fermentation of the deposited strain initially sown on environment, preferably consisting of (by weight):

solution from soaking
to kurusa 1%-4%
agent preventing
foamingonly to
to avoid the formation of foam
- waterup to 100%
- NaOHenough to
bring the pH to about
pH 3.0 to 6.0 before
sterilization environment

The temperature of incubation 27°-36°

Fermentation medium preferably has the following composition (by weight):

the solution from soaking
corn0-4,0%
- downloaded and submitted
cellulose0,8-14%
- Sa Sol0-0,8%
- Ammonium sulphate0-1,0%
agent preventing
foamingonly to
to avoid the formation of foam
- waterenough to
achieve 100%
- NaOHenough to
bring the pH to
about pH 3.0 to 6.0
before sterilization environment
- H2SO4enough to
maintain pH
about 3,0-6,0
- Ammonia (gaseous or
liquid)enough to
maintain pH
about 3,0-6,0

The temperature of incubation 27°-36°C.

For fermentation download the fermenter sufficient amount of water, add ingredients to water in a suitable container with a device for mixing, mix up until ingredients are dissolved. Sterilized, sealing the fermenter and increasing the temperature of the contents is usually to 121°C. the Contents of the fermenter inoculant the seed fermentation medium.

The main source of carbon, which is added in the fermentation process is cellulose; from various sources of cellulose authors prefer to use ARBOCEL, SOLKAFLOC CLAROCEL, ALPHACEL, FIBRACEL different degrees of purity.

The pH in the fermentation process is preferably controlled by adding sulfuric acid or another key is to lot and ammonia in gaseous or liquid form, or other basis.

At the end of the fermentation time, the solids are removed by filtration or centrifugation, collect the liquid phase and concentrate, for example, by ultrafiltration or by organic or mineral membranes.

These enzymes can also be obtained using recombinant DNA techniques, and thus they will be produced recombinant homologous or heterologous species. The host for transfer of the gene encoding the enzyme can be selected from species of fungi, bacterial cells or plant cells. To embed a gene encoding an enzyme in these cells are the owners of both plasmids (integrated or not), phage vectors and vectors of viruses, you can use any convenient means. Penicillum funiculosum, including the insertion of heterologous genes or modification of the genome homologous genes due to inserts, divisions or modifications specified homologous gene, are also part of the present invention.

In accordance with the present invention it is possible to obtain the enzyme in the form of a dedicated pure preparation of the enzyme or in the form of the crude drug, such as culture medium, in which grew Penicillum funiculosum.

You can also enable this enzyme or these enzymes in the composition, containing one enzyme, the type of which depends on the respect to emaho use of the composition. Added enzymes can be, for example, from karbohidrat, lipases and proteases.

C. composition consisting of a mixture of enzyme activities"

1. Liquid composition

For the liquid composition after the addition of antimicrobial agents carry out the measurement of the concentration of enzymes and dilution to the desired concentration. The preferred composition of the liquid solution (by weight) is as follows:

- Microbial products, as all
organic solids4%-10%
- Antimicrobial agent0,005%-0,35%
preferably, the0,01%-0,25%
- Sorbitol20%-50%
- Optional antifreeze0-40%
more preferably15%-40%
- Concentrated filtered
fermentation broth0.3 to 76%

Buffered with bringing the pH to 3-5

Antimicrobial agent is selected from products such as sorbic acid and its salts, benzoic acid and its salts, methyl-4-hydroxybenzoate and n-propyl-4-hydroxybenzoate, fumaric acid, salts and esters. You can also use salt such as sodium chloride is whether potassium chloride.

The preferred cooling agents are 1,2-propandiol, ethylene glycol, glycerin.

2. Powder composition

To obtain powder preparations obtained concentrated solution is dried and optionally in the presence of the media. The powder obtained by drying the concentrated solution without the presence of the media can then be mixed with a suitable carrier.

The preferred composition of the powder form of the composition is the following:

- Microbial product in the form of all
organic solids16%-40%
- Media59%-83%
- Other dried components
fermentation broth1%

Preferred carriers are selected from wheat flour, starch, gypsum, maltodextrin, solid products, corn, by-products derived during the processing of cereals, such as corn grits, second-grade wheat, wheat bran, waste in the processing of rice, a mixture of minerals.

D. characteristics of the enzymes

The new mixture of enzymes produced by Penicillum funiculosum. This mix farm is tov contains new enzymes such as cellulase, β-glucanase, xylanase, such related xylanase enzymes, as arabinofuranoside and feruloyl-esterase.

1. Procedure

The enzyme preparation is described in various analyses, including analyses of the activities of cellulase, cellobiohydrolase, β-glucosidase, endo-1,3(4)-β-glucanase, laminarinase-endo-1,4-β-xylanase (various substrates), β-xyloside, arabinofuranoside and feruloylated (using different substrates).

1.1. DNS CMC method of analysis of cellulase

Analysis of the activity of cellulase is based on the enzymatic hydrolysis of glycosidic linkages in the carboxymethyl cellulose (CMC), β-1,4-glucane. The reaction products, oligosaccharides β-1,4-glucan, determined by the increase in the level of recovery (as glucose).

A solution containing 1 ml of 1% (weight/volume) solution of CMC in 0.1 M nitroacetate buffer, pH 5.0 (or other pH values); 1 ml of appropriately diluted enzyme solution incubated at 50°C for 10 minutes. The enzymatic reaction is stopped by adding 2 ml of DNS solution (1% (weight/ volume)of 3,5-dinitrosalicylic acid, 16% (weight/volume) sodium hydroxide, 30% (weight/volume) potassium sodium (+)-tartrate in distilled water). This solution is stirred and placed in a bath of boiling water (at least 95° (C) for 5 minutes, then Hledat up to 25° C. To the solution was added 10 ml of distilled water, and the absorption was measured at 540 nm using a glass cuvette of thickness 2 cm

The result is transformed into mcmole restored sugar (as glucose)by comparing with a standard curve for 2 ml of 0.00 to 0.04% (weight/volume) solution of glucose treated similarly DNS solution.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the DNS solution is added to the mixture before adding the enzyme solution. One unit of cellulase activity is defined as the amount of enzyme that produces 1 µmol of glucose equivalent per minute under the conditions of the assay (50°C and pH 5.0 or at other pH values).

1.2 Analysis of cellobiohydrolase using p-nitrophenyl β-D-cellobioside

Analysis of cellobiohydrolase based on the enzymatic hydrolysis of p-nitrophenyl β-D-cellobioside. The reaction product, p-NITROPHENOL, determine colorimetrically.

A solution containing 1 ml of 0.1% (weight/volume) of p-nitrophenyl β-D-cellobioside in distilled water; 1 ml of distilled water; 1 ml of 0.2 M nitroacetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution incubated at 50°C for 30 minutes. The enzymatic reaction octanal who live, adding 4 ml of a 0.4 M solution of glycine. This solution is stirred and cooled to 20°C. Absorption was measured at 400 nm using a glass cuvette of thickness 1 cm

The result is transformed into mcmole p-NITROPHENOL, comparing with the molar extinction coefficient of p-NITROPHENOL in the same conditions.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the solution of glycine added to the mixture before adding the enzyme solution. One unit of activity cellobiohydrolase defined as the amount of enzyme that produces 1 µmol of p-NITROPHENOL from p-nitrophenyl β-D-cellobioside per minute under the conditions of the assay (50°C and pH 5.0).

1.3. Analysis β-glucosidase using p-nitrophenyl β-D-glucopyranoside

Analysis β-glucosidase based on the enzymatic hydrolysis of p-nitrophenyl β-D-glucopyranoside. The reaction product, p-NITROPHENOL, determine colorimetrically.

A solution containing 1 ml of 0.1% (weight/volume) of p-nitrophenyl β-D-glucopyranoside in distilled water; 1 ml of distilled water; 1 ml of 0.2 M nitroacetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution incubated at 50°C for 30 minutes. The enzymatic reaction is stopped by adding 4 ml of a 0.4 M solution of the glycine. This solution is stirred and cooled to 20°C. Absorption was measured at 400 nm using a glass cuvette of thickness 1 cm

The result is transformed into mcmole p-NITROPHENOL, comparing with the molar extinction coefficient of p-NITROPHENOL in the same conditions.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the solution of glycine added to the mixture before adding the enzyme solution. One unit of activity β-glucosidase is defined as the amount of enzyme that produces 1 µmol of p-NITROPHENOL from p-nitrophenyl β-D-glucopyranoside per minute under the conditions of the assay (50°C and pH 5.0).

1.4. Analysis of endo-1,3(4)-β-glucanase using DNS and β-glucan barley

Analysis of the activity of endo-1,3(4)-β-glucanase based on enzymatic hydrolysis of glycosidic linkages in β-glucane barley, β-1,3(4)-glucane. The reaction products, oligosaccharides β-1,3(4)-glucan, is determined by the final increase in the level of recovery (as glucose).

A solution containing 1 ml of 1% (weight/volume) solution β-glucan barley in 0.1 M nitroacetate buffer, pH 5.0 (or other pH values); 1 ml of appropriately diluted enzyme solution incubated at 50°C for 10 minutes. The enzymatic reaction is stopped by adding 2 ml of DNS solution (1% (weight/volume)of 3,5-dinitrosalicylic acid, 1,6% (weight/volume) sodium hydroxide, 30% (weight/volume) potassium sodium (+)-tartrate in distilled water). This solution is stirred and placed in a bath of boiling water (at least 95° (C) for 5 minutes, then cooled to 25°C. To the solution was added 10 ml of distilled water, and the absorption was measured at 540 nm using a glass cuvette of thickness 2 cm

The result is transformed into mcmole restored sugar (as glucose)by comparing with a standard curve for 2 ml of 0.00 to 0.04% (weight/volume) solution of glucose treated similarly DNS solution.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the DNS solution is added to the mixture before adding the enzyme solution. One unit activity of endo-1,3(4)-β-glucanase is defined as the amount of enzyme that produces 1 µmol of glucose equivalent per minute under the conditions of the assay (50°C and pH 5.0 or at other pH values).

1.5. Analysis of endo-1,3(4)-β-glucanase using β-glucan azo-barley

Analysis of the activity of endo-1,3(4)-β-glucanase based on enzymatic hydrolysis β-glucans of barley, which has linked chromophore (β-glucan azo-barley). The reaction products, oligomers, which are soluble after precipitation with ethanol, determined according to ludema increase in the absorption at 590 nm.

A solution containing 0.5 ml of substrate β-glucan azo-barley (in a form ready for use) and 0.2 ml of the dilution of the enzyme (containing from 0.15 to 0.60 IU/ml in the CMC in 0.01 M nitroacetate buffer, pH 4,6), incubated at 30°With just over 20 minutes. The enzymatic reaction is stopped by adding 2.5 ml of precipitating solution (containing of 18.1 g of sodium acetate and 3.0 g of zinc mixture, stirred in 300 ml beaker with distilled water, pH adjusted to 5.0 with hydrochloric acid, transfer the contents into a 1 l volumetric flask and bring up to the desired volume of 96% (volume/volume) ethanol). The solution is stirred and left at room temperature for 10 minutes. The solution is transferred into centrifuge vials and centrifuged at 1000 l for 10 minutes in a laboratory centrifuge. The absorption of the supernatant was measured at 590 nm using a glass cuvette of thickness 1 cm

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the precipitating solution was added to the mixture before adding the enzyme solution. One unit activity of endo-1,3(4)-β-glucanase is defined as the amount of enzyme that hydrolyzes the substrate, providing the magnitude of the absorption 0,820 units at 590 nm using a standard substrate in the conditions of the analysis (30°and a pH of 4.6).

1.6. Analysis laminarinase endo-1,3-β -glucanase) using DNS and laminarin

Analysis activity laminarinase (endo-1,3-β-glucanase) is based on the enzymatic hydrolysis of glycosidic linkages in laminaria, β-1,3-glucane. The reaction products, oligosaccharides β-1,3-glucan, is determined by the final increase in the level of recovery (as glucose).

A solution containing 1 ml of 1% (weight/volume) solution of laminarin in 0.1 M nitroacetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution incubated at 50°C for 10 minutes. The enzymatic reaction is stopped by adding 2 ml of DNS solution (1% (weight/volume)of 3,5-dinitro-salicylic acid, and 1.6% (weight/volume) sodium hydroxide, 30% (weight/volume) potassium sodium (+)-tartrate in distilled water). This solution is stirred and placed in a bath of boiling water (at least 95° (C) for 5 minutes, then cooled to 25°C. To the solution was added 10 ml of distilled water, and the absorption was measured at 540 nm using a glass cuvette of thickness 2 cm

The result is transformed into mcmole restored sugar (as glucose)by comparing with a standard curve for 2 ml of 0.00 to 0.04% (weight/volume) solution of glucose treated similarly DNS solution.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in the cat is Roy DNS solution was added to the mixture before adding the enzyme solution. One unit of activity laminarinase defined as the amount of enzyme that produces 1 µmol of glucose equivalent per minute under the conditions of the assay (50°C and pH 5.0).

1.7. Analysis of endo-1,4-β-xylanase using DNA and xylan birch wood

Analysis of the activity of endo-1,4-β-xylanase-based enzyme hydrolysis xyloside linkages in xylan birch wood, β-1,4-xylan. The reaction products, oligosaccharides β-1,4-xylan, determine the ultimate increase in the level of recovery (as xylose).

A solution containing 1 ml of 1% (weight/volume) solution of xylan birch wood in 0.1 M nitroacetate buffer, pH 5.0 (or other pH values); 1 ml of appropriately diluted enzyme solution incubated at 50°C for 10 minutes. The enzymatic reaction is stopped by adding 2 ml of DNS solution (1% (weight/volume)of 3,5-dinitrosalicylic acid, 1,6% (weight/volume) sodium hydroxide, 30% (weight/volume) potassium sodium (+)-tartrate in distilled water). This solution is stirred and placed in a bath of boiling water (at least 95° (C) for 5 minutes, then cooled to 25°C. To the solution was added 10 ml of distilled water, and the absorption was measured at 540 nm using a glass cuvette of thickness 2 cm

The result is transformed into mcmole restored sugar (as xylose), comparing with the standard curve for 2 ml 0,00-0,03% (weight/volume) solution of xylose, processed similarly DNS solution.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the DNS solution is added to the mixture before adding the enzyme solution. One unit activity of endo-1,4-β-xylanase is defined as the amount of enzyme that produces 1 µmol xyloskalo equivalent per minute under the conditions of the assay (50°C and pH 5.0 or at other pH values).

1.8. Analysis of endo-1,4-β-xylanase using DNS and arabinoxylan wheat

Analysis of the activity of endo-1,4-β-xylanase-based enzyme hydrolysis xyloside ties in arabinoxylan wheat, arabinose substituted β-1,4-xylan. The reaction products, oligosaccharides, arabino-β-1,4-xylan, determine the ultimate increase in the level of recovery (as xylose).

A solution containing 1 ml of 1% (weight/volume) solution of arabinoxylan in 0.1 M nitroacetate buffer, pH 5.0 (or other pH values); 1 ml of appropriately diluted enzyme solution incubated at 50°C for 10 minutes. The enzymatic reaction is stopped by adding 2 ml of DNS solution (1% (weight/volume)of 3,5-dinitrosalicylic acid, 1,6% (weight/volume) sodium hydroxide, 30% (weight/volume) potassium sodium (+)-tartrate in distilled water). This solution manual is more and placed into a bath of boiling water (at least 95° C) for 5 minutes, then cooled to 25°C. To the solution was added 10 ml of distilled water, and the absorption was measured at 540 nm using a glass cuvette of thickness 2 cm

The result is transformed into mcmole restored sugar (xylose), comparing with the standard curve for 2 ml 0,00-0,03% (weight/volume) solution of xylose treated similarly DNS solution.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the DNS solution is added to the mixture before adding the enzyme solution. One unit activity of endo-1,4-β-xylanase is defined as the amount of enzyme that produces 1 µmol xyloskalo equivalent per minute under the conditions of the assay (50°C and pH 5.0 or at other pH values).

1.9. Analysis of endo-1,4-β-xylanase by viscometric method using arabinoxylan wheat

Analysis of the activity of endo-1,4-β-xylanase-based enzyme hydrolysis of a standard solution of arabinoxylan wheat, and the activity determined by the decrease in relative viscosity with time.

A solution containing 1 ml of 1% (weight/volume) solution of arabinoxylan wheat in 0.1 M nitroacetate buffer, pH 5.5 (or other pH values); 3 ml of distilled water and 1 ml correspond to the way the diluted enzyme solution is injected into microviscosity Haake (using a gold ball calibrated to 0.1-2.0 MPa·s), and measure the time of fall of the ball (Ttest(in msec with respect to a particular drop height) every 30 sec for 15-20 minutes at 30°C. the Average time of fall of the ball is measured for water (5 ml distilled water) and substrate (1 ml of 1% (weight/volume) solution of arabinoxylan wheat in 0.1 M nitroacetate buffer, pH 5.5, and 4 ml of distilled water) as Twaterand Tsubstrate, respectively. Conduct test measurements in a similar way. The relative fluidity (Fr) compute for each value of Ttestas follows:

The slope of the curve Frdepending on time (elapsed time, which held each dimension Ttest), calculated as the change in relative viscosity per minute (ΔFr/min), and it is proportional to the concentration of the enzyme.

One unit activity of endo-1,4-β-xylanase is defined as the amount of enzyme that hydrolyzes the substrate, reducing the viscosity of the solution, providing the change in relative yield 1 (dimensionless unit) per minute under the conditions of analysis (30°C and pH 5.5, or at other pH values).

1.10. Analysis β-xyloside using p-nitrophenyl-β-xylopyranoside

Analysis β-xyloside based on ferment the m hydrolysis of p-nitrophenyl β -D-xylopyranoside. The reaction product, p-NITROPHENOL, determine colorimetrically.

A solution containing 1 ml of 0.1% (weight/volume) of p-nitrophenyl-β-D-xylopyranoside in distilled water; 1 ml of distilled water; 1 ml of 0.2 M nitroacetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution incubated at 50°C for 30 minutes. The enzymatic reaction is stopped by adding 4 ml of a 0.4 M solution of glycine. This solution is stirred and cooled to 20°C. Absorption was measured at 400 nm using a glass cuvette of thickness 1 cm

The result is transformed into mcmole p-NITROPHENOL, comparing with the molar extinction coefficient of p-NITROPHENOL in the same conditions.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the solution of glycine added to the mixture before adding the enzyme solution. One unit of activity xyloside defined as the amount of enzyme that produces 1 µmol of p-NITROPHENOL from p-nitrophenyl-β-D-xylopyranoside per minute under the conditions of the assay (50°C and pH 5.0).

1.11. Analysis α-N-arabinofuranoside using p-nitrophenyl α-L-arabinofuranoside

Analysis α-N-arabinofuranoside (arabinofuranoside) based on the enzymatic hydrolysis of p-nitrophen the l α -L-arabinofuranoside. The reaction product, p-NITROPHENOL, determine colorimetrically.

A solution containing 1 ml of 0.1% (weight/volume) of p-nitrophenyl α-L-arabinofuranoside in distilled water; 1 ml of distilled water; 1 ml of 0.2 M nitroacetate buffer, pH 5.0; 1 ml of appropriately diluted enzyme solution incubated at 50°C for 30 minutes. The enzymatic reaction is stopped by adding 4 ml of a 0.4 M solution of glycine. This solution is stirred and cooled to 20°C. Absorption was measured at 400 nm using a glass cuvette of thickness 1 cm

The result is transformed into mcmole p-NITROPHENOL, comparing with the molar extinction coefficient of p-NITROPHENOL in the same conditions.

In the observed absorption for solutions of enzyme reactions make a correction for nonspecific absorption, carrying out the reaction in which the solution of glycine added to the mixture before adding the enzyme solution. One unit of activity arabinofuranoside defined as the amount of enzyme that produces 1 µmol of p-NITROPHENOL from p-nitrophenyl α-L-arabinofuranoside per minute under the conditions of the assay (50°C and pH 5.0).

1.12. Analia feruloyl-esterase FAXX method

Analysis feruloyl-esterase (esterase ferulic acid) based on the enzymatic hydrolysis of O-[5-O-(TRANS-feruloyl)-α-L-arabinofuranosyl]-(1Ȓ 3)-O-β-D-xylopyranosyl-(1→4)-D-xylopyranose (FAXX). FAXX get from enzymatically hydrolyzed wheat bran, purified and characterized by NMR. FAXX hydrolysis determined spectrophotometrically.

The enzymatic reaction observe at a wavelength of 325 nm using a cuvette with 1 cm thickness, the solution containing 0,050 mm FAXX in 0.1 M MOPS buffer, pH 6.0 at 37°C.

One unit of activity feruloyl-esterase on FAXX is defined as the amount of enzyme that converts 1 µmol of substrate to product per minute under the conditions of analysis (37°C and pH 6.0).

1.13. Analysis feruloyl-esterase using Ara2F

Analysis feruloyl-esterase (esterase ferulic acid) based on the enzymatic hydrolysis of Ara2F (ferulic acid linked by 1,2 with arabinose). Ara2F is obtained from enzymatic hydrolyzed pulp sugar beet, purified and characterized by NMR. Ara2F hydrolysis determined spectrophotometrically.

Enzymatic reactions observed at a wavelength of 325 nm using a cuvette with 1 cm thickness, the solution containing 0,050 mm Ara2F in 0.1 M MOPS buffer, pH 6.0 at 37°C.

One unit of activity feruloyl-esterase on Ara2F is defined as the amount of enzyme that converts 1 µmol of substrate to product per minute under the conditions of analysis (37°C and pH 6.0).

1.14. Analysis feruloyl-esterase with what ispolzovaniem hydrolysis of methyl esters: metiferonului acid (MFA); methylcathinone acid (MSA); medicinova acid (MSA); methyl-p-coumaric acid (MpCA)

Analysis feruloyl-esterase (esterase ferulic acid) based on the enzymatic hydrolysis of methyl esters of ferulic acid (MFA); caffeic acid (MSA); sinoway acid (MSA); and p-coumaric acid (MRSA). Hydrolysis of methyl esters determined in 0.1 M MOPS buffer, pH 6.0 at 37°C. the Analysis is based on two different methods.

In the spectrophotometric method, the substrate concentration of methyl esters is 0.10 mm, and for the hydrolysis of esters see at a wavelength of 325 nm using a cuvette with a thickness of 1 see In this way the initial concentration of substrate is limited.

In the method using HPLC the substrate concentration of methyl esters is 1.0 mm, and for the hydrolysis of esters is monitored by measuring the allocation of free acid by HPLC with 10-30 minute intervals. In this way there is no limit on the concentration of the substrate, and the measured activity is much higher than the concentration determined spectrophotometrically.

One unit of activity feruloyl-esterase is defined as the amount of enzyme that converts 1 µmol of substrate to product per minute under the conditions of analysis (37°C and pH 6.0).

1.15. Determination of protein concentration using the option the sale analysis Bradford binding protein with Coomassie blue

The analysis of the concentration of protein is based on a modified Bradford analysis of the binding protein with Coomassie blue using Brilliant Blue G (Kumasi diamond blue), measured spectrophotometrically at a wavelength of 595 nm using a cuvette with a thickness of 1 see This method (Sigma 6916) standardized using bovine serum albumin (Sigma P094).

1.16. Determination of isoelectric point using isoelectric focusing

Isoelectric point of proteins determined by standard methods using pre-cast vertical 5% polyacrylamide gels, such as gels from NOVEX® for the range of pH 3-10 (P1 interval 3,5-8,5) or pH 3-7 (P1 interval of 3.0 to 6.5) in a ditch NOVEX® XCell II™ Mini-Cell. Use NOVEX® the cathode, the anode and IFE exemplary buffers for pH 3-10 or pH 3-7. Use NOVEX® a standard Protocol for isoelectric focusing, fixation, staining dye Coomassie R-250 Blue and desalination.

1.17. SDS-PAGE (gel electrophoresis in nitrilotriacetate polyacrylamide gel)

Analytical separation and molecular weight determination of proteins carry out standard SDS-PAGE methods. Pre-cast NOVEX® NuPAGE gels (NuPAGE™ Bis-Tris gels and NuPAGE™ Tris-acetate gels from NOVEX® recommended working buffers used in NOVEX® XCell II™ Mini-Cell. Use NOVEX® education is zowie preparative and working buffers and standards of molecular weights. Use NOVEX® standard protocols for SDS-PAGE, fixation, staining dye Coomassie R-250 Blue and desalination.

2. The results of the analysis of a mixture of enzymes

2.1. The optimum pH values

2.1.1. The activity of endo-1,3(4)-β-glucanase

Analysis of endo-1,3(4)-β-glucanase from Penicillum funiculosum carried out under standard conditions at 50°With, by using a method using DNS and β-glucan barley. Enzymatic activity was measured at pH values between a 3.0 and 7.0. Optimum pH for activity of the enzyme consists of 4.0 to 5.0.

pHActivity
the Intern. u/min(%)
332542
476198
5775100
650766
715220

2.1.2. The activity of endo-1,4-β-xylanase

Analysis of endo-1,4-β-xylanase from Penicillum funiculosum carried out under standard conditions at 50°With, by using a method using DNS and xylan birch wood.

pHActivity
(proc. u/min) (%)
123
2,0355937
2,6670070
3,0841188
3,0811385
3,59582100
4,0852389
4,0851089
5,0554458
5,5352237
Continuation of the table.
123
6,0219023
7,0110312

2.2. The optimum temperature

2.2.2. The activity of endo-1,3(4)-β-glucanase

Analysis of endo-1,3(4)-β-glucanase from Penicillum funiculosum carried out under standard conditions at pH 5.0 (the optimal pH for this enzyme) by way of using DNS and β-glucan barley. Enzymatic activity was measured at temperatures between 30 and 70°C. the Values of the optimal temperatures are between 50 and 60°With the highest activity observed at 60°C. Detailed results in the form of a table depending on temperature Pref is found in table

TemperatureActivity
(proc. u/min)(%)
3024732
4054170
50775100
601082140
7077496

2.2.2. The activity of endo-1,4-β-xylanase

Analysis of endo-1,4-β-xylanase from Penicillum funiculosum carried out under standard conditions at pH 5.5 and 3.5, with the method using the DNS and xylan birch wood.

Enzymatic activity was measured at temperatures between 30 and 70°C. the Values of the optimal temperatures are between 50 and 60°With the highest activity observed at 50°for pH 5.5 and 60°for pH 3.5. Detailed results in the form of a table depending on the temperatures shown in the table

Temperature (°)Activity (pH 5.5)Activity (pH 3.5)
(Med/min)(%)(Med/min)(%)
30249241433423
40404266812842
5061071001825195
6046027519155100
703851631273066

Enzymes produced by Penicillum funiculosum, have high levels of cellulase, endo-1,3(4)-β-glucanase and other glycolytically activities. In addition, they are also characterised by high levels of endo-1,4-β-xylanase and activities of related enzymes xylanase. A wide range of hemicellulolytic enzymes is characteristic of enzyme preparations derived from this organism.

Each of the measured activities can be represented as a relation to the main activity of this drug. Examples of obtained results are presented in table A. These relationships can vary in preparations obtained from different batches of fermentation.

Table a: Relative activity depending on various substrates
The methods used for testingResults for Penicillum funiculosum
1 2
Cellulase system (DNS CMC method, pH 5.0) [1.1]3,14
Cellobiohydrolase (p-nitrophenyl β-D-cellobioside way, pH 5.0) [1.2]0,022
β-glucosidase (p-nitrophenyl β-D-glycobiotechnology way, pH 5.0) [1.3]of) 0.157
Endo-1,3(4)-β-glucanase (the way DNS and β-glucan barley, pH 5.0)[1.4]of 7.23
Endo-1,3(4)-β-glucanase (way β-glucan azo-barley, pH 4,6) [1.5]1+/-
Laminarinase (the way DNS and laminarin, pH 5.0)[1.6]0,30
Endo-1,4-β-xylanase (the way DNS and xylan birch wood, pH 3.5) [1.7]9,16
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat, pH 3.5) [1.8]8,67
Endo-1,4-β-xylanase (viscometric method of arabinoxylan wheat, pH 5.5) [1.9]9,80
Continuation of the table And
12
β-xyloside (method p-nitrophenyl-β-D-xenobiotica is the Zid) [1.10] 0,0047
α-N-arabinofuranoside (method p-nitrophenyl-α-L-arabinofuranoside) [1.11]0,0017
Feruloyl-esterase (FAXX) [1.12]0,000254
Feruloyl-esterase (Ara2F) [1.13]0,000349
Feruloyl-esterase (MFA spectrophotometric method) [1.14]0,000135
Feruloyl-esterase (ACI spectrophotometric method) [1.14]0,000174
Feruloyl-esterase (MSA spectrophotometric method) [1.14]0,000049
Feruloyl-esterase (MRSA spectrophotometric method) [1.14]0,000216

3. Properties of components in the mixture of enzymes

3.1 cleaning Methods

Chromatography hydrophobic interactions

Preparations obtained after filtration and concentration of the fermentation medium to a protein concentration of 112,6 mg/ml, diluted 1/1 buffer for chromatography hydrophobic interaction (HIC) (50 mm phosphate buffer, pH 7.0/1.7 M (NH4)2SO4/0,04% sodium azide), replace with HIC buffer (PD-10 column; Pharmacia). Portions (10 ml) high-efficiency HIC gel (Pharmacia) enter in column (diameter 10×5 cm, 200 ml) PhenylSepharoes™and section is Aut, using a gradient of ammonium sulfate (NH4)2SO4with decreasing concentration (1,7-0,0 M) for 10 minutes. Fractions (10 ml) are collected and examined for the activity of xylanase.

HIC provides two major peaks of activity of xylanase. The first, called A, eluted from the column when the concentration of (NH4)2SO4is reduced to about 0.6 M, while the second, named, eluted at a concentration of (NH4)2SO4about 0.25 M. the Fractions containing the peaks a and b, from each input collected separately. Full fraction And corresponds to 2.8% of the total xylanase activity, whereas the fraction corresponds In 97.2% of the full activity of xylanase. The output is 77%.

Ion-exchange chromatography

Combined fractions of peaks a and b of the HIC precipitated by increasing the concentration of (NH4)2SO4to 100% saturation, followed by centrifugation (10000×g for 30 minutes). Precipitation again dissolved in a mixture of 20 ml of Tris-HCl buffer, pH 8.0/0.04% of sodium azide and absoluut to the same buffer using a PD-10 column. Samples (5 ml) is injected into MonoQ™ HR 10/10 anion exchange column (Pharmacia), previously equilibrated with a mixture of 20 mm Tris-HCl buffer pH 8.0/0,04% sodium azide, and elute at a rate of 2 ml/min with increasing concentrations of sodium chloride (NaCl(0-1 M) in the same buffer. Fractions (2 ml) to collect and analyze activity xila the basics.

Peak And:

The selection of the peak And using anion-exchange chromatography results in one maximum xylanase activity, which eluted at 0.3 M NaCl. The most active fractions are collected and analyzed by SDS-PAGE (gel electrophoresis in nitrilotriacetate the polyacrylamide). Get one major band with a molecular mass of 48 kDa. The selection activity of xylanase after IEF (isoelectric focusing) confirms that this major Coomassie-stained band is a xylanase.

Peak:

The selection of the peak In using anion-exchange chromatography gives two major peaks of activity of xylanase, one of which eluted in the peak nesorbiruyushchegosya material (unbound material; peak B-I)and the other with 0.1 M NaCl (peak II). There are also two small peaks, which suiryudan at 0.13 and to 0.19 M NaCl. Active fractions corresponding to each peak is collected and analyzed by SDS-PAGE, but none of the samples is not clear.

Gel-filtration chromatography

Combined fractions containing In-I and-II, is dried by freezing, again dissolved in water and absoluut (using PD-10 columns). Samples (0.2 ml) is injected into Superdex™ 75 HR column (Pharmacia) and elute at a speed of 0.4 ml/min buffer 20 mm Bis-Tris, pH 6.0/0.2 M NaCl/0,04% sodium azide. Fractions (0.4 ml) to collect and analyze on xylanase activity.

3.2 Characteristics of xylanase

3.2.1. Determination of isoelectric point using isoelectric focusing

Isoelectric point of proteins determined by standard methods using pre-cast vertical 5% polyacrylamide gels from NOVEX® for pH 3-10 and pH 3-7. Use NOVEX® the cathode, the anode and IEF exemplary buffers, and standard protocols for isoelectric focusing, fixation, staining Coomassie R-250 Blue dye, and discoloration.

For xylanase And use the sample after MonoQ. For xylanases In-I and-II use the sample after HlC, xylanase Century For each of a and b a small sample (10 µl) was placed in a single cell, and a large sample (50 μl) was placed in a triple cell. After focusing the samples, the gel is cut into two parts so that one part contains two small sample (a+b) and molecular weight markers (this half of the Coomassie stained), while the other part contains two large sample. Part of the gel, which contains large samples, cut so as to divide the strips of the two samples, and then each band is divided into sections of 2 mm Each of 2 mm sections soaked separately overnight in 100 mm MOPS buffer, pH 6.0/0.04% of sodium azide. Fractions examined for the activity of xylanase.

For sample And xylanase stained IEF gel demonstrates one baseband pI 3,55 marker and a few small is their impurity bands. Silananda activity was detected only in fractions corresponding to this band, confirming that the main peak corresponds to the xylanase.

For sample stained IEF gel shows several bands in the range of pI values. Silananda activity was separated in two fractions unpainted gel and corresponds to proteins with a pI of 4.2 and 4.8.

3.2.2 Determination of molecular weight by SDS-PAGE

To confirm the molecular mass of xylanase in the peak of HIC fractions with xylanase activity, elyuirovaniya of the IEF gel, absoluut, dried by freezing and separated by SDS-PAGE. Denaturing PAGE carried out using 10% Tris-glycine gel (NOVEX®) dithiothreitol (50 mm DTT), included in the exemplary buffer as a reducing agent.

Stained gel shows that both xylanase was clean, with a molecular mass of 36 kDa and 15 kDa for xylanase In-I and-II, respectively.

All three purified xylanase analyzed by SDS-PAGE: the fraction of xylanase And after anion-exchange chromatographic processing, the fraction of xylanase In-I and-II after gel-filtration chromatographic processing. Xylanase And gives a single band with a molecular mass of 48 kDa. Xylanase B-I gives one major and four minor bands after Coomassie staining. Confirmed that the main band to meet the t of the xylanase, because of its molecular mass of 36 kDa. The degree of purity is estimated as 90%. Xylanase-II gives the main band with a molecular mass of 15 kDa and 2-3 small strips. The degree of purity of this xylanase approximately 95%.

SampleM mass (kDa)P.I.
Xylanase And483,55
Xylanase B-I364,20
Xylanase B-II154,80

3.2.3. Enzyme activity

Tests for the measurement of enzyme activity have been described above.

3.2.3.1. Analysis of xylanase And

[Protein] of 0.4 (mg/ml)

The method of analysis of enzymepHThe enzyme activity
(IU/ml)(u/mg protein)
1234
Cellulase system (DNS CMC method) [1.1]5,0<1,0N/a
Cellobiohydrolase (p-nitrophenyl β-D-cellobioside method)[1.2] 5,0<1,0N/a
Endo-1,3(4)-β-glucanase (the way DNS and β-glucan barley) [1.4]5,0<1,0N/a
Continuation of the table
1234
Laminarinase (the way DNS and laminarin) [1.6]5,0butN/a
Endo-1,4-β-xylanase (the way DNS and xylan birch wood) [1.7]5,5140350
Endo-1,4-β-xylanase (the way DNS and xylan birch wood) [1.7]3,5158395
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat) [1.8]5,5152380
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat) [1.8]3,5171429
Endo-1,4-β-xylanase (viscometric pic is b arabinoxylan wheat) [1.9] 5,54561140

But - not determined

N/a - not applicable

The xylanase activity against xylan birch wood depending on pH

pHThe enzyme activity
(Proc. u/mg protein)(% of maximal activity)
2,0029473
3,0035387
3,5038595
4,00405100
5,0034585
5,5034084
6,0030275
7,0021252

3.2.3.2. Analysis of xylanase In-I

[Protein] 0,096 (mg/ml)

The method of analysis of enzymepHThe enzyme activity
(IU/ml)(u/mg protein)
1234
Cellulase system (DNS CMC method) [1.1]5,026,5276
Cellobiohydrolase (p-nitrophenyl β-D-cellobioside method) [1.2]5,00,5415,6

Continuation of the table
1234
Endo-1,3(4)-β-glucanase (the way DNS and β-glucan barley) [1.4]5,073,8769
Laminarinase (the way DNS and laminarin) [1.6]5,0<0,1N/a
Endo-1,4-β-xylanase (the way DNS and xylan birch wood) [1.7]5,551,3534
Endo-1,4-β-xylanase (the way DNS and xylan birch wood) [1.7]3,583,6871
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat) [1.8]5,593,2971
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat) [1.8]3,5143,81498
Endo-1,4-β-xylanase (viscometric method of arabinoxylan wheat) [1.9]5,51471531

But - not determined

N/a - not applicable

The activity of xylanase in respect of xylan, the wood is s birch depending on pH

pHThe enzyme activity
(Proc. u/mg protein)(% of maximal activity)
2,0061070
3,0075587
3,50871100
4,0080292
5,0056765
5,5053461
6,0048155
7,0040446

3.2.3.3. Analysis of xylanase In-II

[Protein] 0,165 (mg/ml)

The method of analysis of enzymepHThe enzyme activity
(IU/ml)(u/mg protein)
1234
Cellulase system (DNS CMC method) [1.1]5,0<1,0N/a
Cellobiohydrolase (p-nitrophenyl β-D-cellbio-paranoidly way [1.2]5,0<0,1N/a

Continuation of the table
1234
Endo-1,3(4)-β-glucanase (the way DNS and β-glucan barley) [1.4]5,0<1,0N/a
Laminarinase (the way DNS and laminarin) [1.6]5,0butN/a
Endo-1,4-β-xylanase (the way DNS and xylan birch wood) [1.7]5,5141,9860
Endo-1,4-β-xylanase (the way DNS and xylan birch wood) [1.7]3,5261,01582
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat) [1.8]5,5152,6925
Endo-1,4-β-xylanase (the way DNS and arabinoxylan wheat) [1.8]3,5267,91624
Endo-1,4-β-xylanase (viscometric method of arabinoxylan wheat) [1.9]5,52621588

But - not determined

N/a - not applicable

The xylanase activity against xylan birch wood depending on pH

pHActive is any enzyme
(Proc. u/mg protein)(% of maximal activity)
2,00137484
3,00152393
3,50158297
4,001630100
5,00109367
5,5086053
6,0044327
7,0015610

3.2.4 Sequence

One of the variants of the present invention relates to the amino acid sequence and nucleic acids for the above proteins or their variants.

For this purpose, the sequence of the xylanase identify amino acid sequences of the purified protein (xylanase And xylanase In-I and xylanase B-II) and the comparison amino acid sequence and nucleotide sequences of known fungal xylanases.

It should be understood that in the framework of the present invention, the term options refers to any polypeptide or any similar protein, protein fragment, derivative or protein mutant of the native protein or polypeptide and having the same biological function as specified native is a protein or polypeptide. In a natural state can exist different ways. Such variants may be, for example, allelic variants, characterized by differences in the sequence of the genes encoding the indicated protein, or can be the result of differential splicing or post-transduction modifications. Options can be obtained by substitution, deletion, addition and/or modification of one or more of the amino acids. All modifications are well known and can be implemented by any known in the art methods.

Options are molecules having, for example, high affinity for their substrate, or with novel biological properties.

Another objective of the present invention is also the use of sequences for the expression of the described proteins or polypeptides in cells-the owners of single-celled or multicellular organisms. For this purpose, these sequences can be introduced into the genome of the vector. The specified vector may be a plasmid, phage or virus. Therefore, another option of the present invention is a host cell, selected from a single-celled or multicellular organism, transfected or infected with the vector, as described above. In a preferred embodiment, the host-cell is a bacterium.

Another variant of this izaberete the Oia is the use of said vectors, comprising a sequence of nucleic acids described proteins for expression of the indicated protein in any cell of the host.

3.2.4.1 Sequence of xylanase From

Creating probes based on comparisons of amino acid and nucleotide sequences of known fungal xylanases. Define conservative areas and used to generate PCR primers, the products of which could be used for screening of the genomic library Penicillum funiculosum.

Create two pairs of degenerate primers. The first couple pose for amplification of a 200 bp (approximately) of the product of the gene ksiezy type b (or type 2). The second pair create to amplify a 250 bp product of the xylanase gene of type a (type 1).

Strip 258 bp obtained using primers 3 and 4. After cloning into pGEMT and sequencing it was found that there is significant sequence similarity with a sequence of a fungal xylanase type a/1. The plasmids containing the cloned product, called pPFXYLA.

The full sequence of xylanase From shown in Fig. 1 and sequence ID No. 1 (AFTERBIRTH. ID No. 1).

3.2.4.2 Sequence of xylanase BI

To design degenerate PCR primers ((AFTERBIRTH. ID No. 2 and No. 3) use an internal amino acid sequence, along with comparisons of the sequences of other fungal whole is of ovigerous. Product 290 bp (AFTERBIRTH. ID No. 4) amplified and clone into pGEMT (Promega) to create pGEMTCB2 and is sequenced. In Fig. 2 shows that primernye sequence are underlined. This PCR product currently used as a probe for screening of the genomic library Penicillum funiculosum IMI134756.

3.2.4.3. The sequence of xylanase BII

The entire sequence of the gene of xylanase BII includes a 1.3 kb 5 netransliruemoi and located in the opposite direction of the plot and to 0.85 kb 3 netransliruemoi plot, 54 bp of the intron and 669 bp encoding a protein of 223 amino acids.

For evidence of the existence of 54 bp of intron using PCR with reverse transcriptase (RT-PCR). Total RNA isolated from mycelium cultures Penicillum funiculosum IMI134756 collected after 4 days of cultivation on 1% (weight/volume) xylan oat. Design primers for the amplification up to a 195 bp fragment of the RNA (249 bp of genomic DNA) and to a 433 bp (487 bp from genomic DNA).

Sequencing of 3 kb 3 konza plasmids (pPFXYNC2) revealed a gene (denoted by per (A), which contains two predicted introns, and encodes a polypeptide consisting of approximately 570 amino acids. This polypeptide shows significant sequence similarity with the amino acid permease fungi.

3.2.4.4. The sequence of xylanase And

Was obtained internal consistency of xylanase A, and it is not only the next sequence of amino acids: AEAINYNQDY

3.3. Properties feruloyl-esterase

3.3.1. Clean

Carry out the same method as for xylanases.

Enzyme blend contains at least two different feruloyl-esterase. One of them (FaeB) molecular weight according to mass spectrometry is 38,945-41,051 Da (35,450 Da from the primary amino acid sequence and 37 kDa according to SDS-PAGE. FaeB has a value of pI of 4.2, which corresponds to feruloyl-esterase type and is specific to MRSA and Ara2F substrates (activity against MRSA, MCA, MFA and Ara2F; but not in respect of the MSA and FAXX).

In another feruloyl-esterase (FaeA) molecular weight is 29 kDa (according to SDS-PAGE. FaeA is set to pI 4,65, which corresponds to feruloyl-esterase and is specific for FAXX and MSA substrates (activity against MSA, MSA, MFA and FAXX, but not in relation to MRSA Ara2F).

3.3.2. Determination of isoelectric point using isoelectric focusing.

Isoelectric point of proteins define standard ways. The mixture of enzymes applied in the form of wide strips (about 20 mm) in the IEF gel and carry out electrophoresis at low temperature (5°). After focusing and narrowing strip of gel cut on the middle strip of the sample. One part of the stripe pattern and IEF standards fixed, stained and absoluut using a standard Protocol. Other the second part of the strip is cut into sections of 2 mm, and each area is soaked overnight in 1 ml of 100 mm MOPS, pH 6.0. Activity feruloyl-esterase determine for each section of the gel, using MFA, MRA and MSA as substrates.

Stained IEF gel shows the presence of many proteins in the cellulase, pI values for which range from very acidic (pI 2,4) to about pI 7. Most proteins are acidic (interval pI 2,4-5). Two peak feruloyl-esterase activity find in fractions, cut from the gel. One corresponding FaeB has a value of pI of 4.2 and activity only in relation to MFA, MRSA (but not MSA). The other, corresponding FaeA has the value of pI 4,65 and activity against all three tested substrates.

3.3.3 Molecular mass determined by SDS-PAGE

Molecular weight determined by SDS-PAGE using 10% Tris-glycine gels. Get SDS-PAGE gels, fixed, stained Coomassie Blue dye and absoluut using a standard Protocol.

Enzyme blend contains at least two different feruloyl-esterase. One of them, the corresponding FaeB (pI 4,2), molecular mass of 37 kDa. The other, corresponding FaeA (pI 4,65), molecular weight is 29 kDa.

The molecular weight of FaeB appreciate how 34,450 Da from the primary amino acid sequence, and as 38,945-41,051 Da according to the mass spectrometer is I.

3.3.4. Activity feruloyl-esterase

Analysis on the activity of feruloyl-esterase carried out on a mixture of enzymes, using spectrophotometric method.

SubstrateActivity
(IU/ml)(U/g protein)
Methyl ferulate MFA (0.1 mm)0,0867,9
Methyl caveat ACI (0.1 mm)0,11110,3
The methyl sinapate MSA (0.1 mm)0,0312,9
Methyl p-camarat MRSA (0.1 mm)was 0.138a 12.7
FAXX (0.05 mm)rate £ 0.16215,0
Ara2F (0.05 mm)0,22220,6

The mixture contains enzymes activity against all tested substrates. For methylesters the highest activity is the activity in relation to MRSA and lowest in relation to the MSA.

Activity against Ara2F and FAXX higher than for methylesterase that is an indication that the activity of esterases associated with the true feruloyl-esterases, and not with the General esterases or by other activities destroying the cell walls of esterases (e.g., acetyl xylan-esterase, pectin-esterase).

3.3.5. After the outermost

3.3.5.1 Sequence FEA-And

In accordance with the splitting of the purified proteins obtain internal amino acid sequence as:

Sequence 1

QYTLTLPSNYNPNK

Sequence 2

AVAVMSGANL

Sequence 3

TEYSG (C/a) DSEHPVWWIAFDGP

Sequence 4

DTFVKDDHCTPTNPPAPAAGSGTHIKYV

Design several degenerate PCR primers from the amino acid sequences obtained from the purified protein. Many products were cloned into pGEMT (Promega) and sequenced.

Using PCR, it was found that plasmid, named pGEMTD19 (180 bp) (figure 3), contains a sequence that is recognized as a peptide sequence 4, above. As shown in figure 3, primernye sequence are underlined twice, and formerly known sequence is underlined once.

Nucleic acid sequence and amino acid FAE-A presents in the PLACENTA. ID No. 5.

3.3.5.2. The sequence FEA-B

The primers, designed from peptide sequences FAE-B, used for amplification of the probe, which is then used for screening of the genomic library Penicillum funiculosum. Clone 2291 bp is isolated and is sequenced (AFTERBIRTH. ID No. 6). The gene encoding the polypeptide of 304 amino acids, has an estimated one intron. The predicted amino acid sequence of the pre is presented in figure 4, where the Mature protein (the length of the Mature protein =338) are shown in bold. This protein consists of two different domain, divided into heavily glycosylated linker. As shown in figure 4, the catalytic domain is shown in bold, whereas binding domain are shown in bold and double-underlined, and the linker are dotted bold line.

Protein also differs alleged fragment of the active site (serine=nucleophile), which is represented by the underline in figure 4, with the following hypothetical catalytic triads:

(1) S136/D174/H216

(2) S136/D220/H276.

FAE-B protein includes a sequence secretion (353) and 10 cysteines.

3.4 properties of the glucanase

The mixture of enzymes is treated using 2D gel electrophoresis. IEF carried out using pre-cast vertical 5% polyacrylamide gels from NOVEX® for pH 3-7 (interval pI 3,0-6,5) in a mini-cell NOVEX® XCell II™ Mini-Cell. Use NOVEX® the cathode, the anode and IEF exemplary buffers for pH 3-7 and NOVEX® a standard Protocol for isoelectric focusing. One strip cut, and electrophoretic process in the second direction using a 10% Laemmli SDS-PAGE gel. From gel emit a second band, cut into 35 fractions strips of the gel is soaked in a buffer, and fractions analyzed for enzyme activity. T the th band is left on the gel, fixed, stained Coomassie R-250 Blue dye and absoluut using NOVEX® standard Protocol.

Significant activity of endo-1,3(4)-β-glucanase (the way DNS and β-glucan barley) and cellulase (the way DNS and CMC) was detected in the fractions corresponding to proteins with a pI of 4.2, M.V. 36 kDa and a pI of 5.4, M.V. 27 kDa. Removal of xylanase In-1, located in one of the fractions, the fractions are tested for activity using the method of the DNS and xylan birch wood. In the fractions corresponding to the activities β-glucanase or cellulase, xylanase activity was not detected.

List of drawings

Figure 1: Amino acid sequence of the protein xylanase From Penicillium funiculosum.

Figure 2: Nucleotide and amino acid sequence of the PCR product xylanase B1 (XynBI).

Figure 3: Nucleotide and amino acid sequence of the PCR product feruloyl-esterase A (faeA).

Figure 4: Amino acid sequence of the protein (FAE-V or FAE-I) feruloyl-esterase (faeB) Penicillium funiculosum

That is, the Application of a mixture of enzymes in animal feed

Example 1: evaluation of the efficacy of enzymes produced by Penicillium funiculosum in relation to the energy value (AMEN) feed from a mixture of wheat and barley for broilers.

The aim was to demonstrate the effectiveness of the enzymes (activity β-glucanase: 100 u/kg and activity of xylanase: 1100 IU/kg) on apparent about the military energy, corrected for nitrogen balance (AMEN) feed containing 50% wheat and 22% barley.

The experiments were conducted using the control and enzyme preparations (activity β-glucanase: 100 u/kg and activity of xylanase: 1100 IU/kg), using the European method of comparison (Bourdillon et al., 1990) when feeding ad libitum and full collection of excreta for ages between 18 and 21 day.

If necessary, the obtained experimental enzyme preparations were supplemented by separate enzymes to obtain the specified enzyme activity of the tested enzyme preparations. When the activity of the party of enzyme preparation on β-glucanase was 70 u/kg, the drug for inclusion in the experiment was supplemented isolated from Penicillium funiculosum and purified enzyme to the desired activity.

A. Material and methods

Birds: the species and growing conditions

Day male Ross broiler chickens kept in cages collective battery until the age of 12 days. They give standard initial feed. On day 12 of the birds are weighed and evenly distribute on 10 separate cells for each treatment, and then give them experimental food during the adaptation period (minimum 5 days).

Support standard temperature and humidity. Constant light regime is: 23 hours light and 1 hour of darkness until the end of the test.

Feed the Birds get the initial feed from one day to 12 days, and then receive the experimental feed.

Experimental feed

Feed containing 50% wheat and 22% barley (table 1.1). The enzyme preparation is sprayed on 20 kg of grain.

Recovery of enzymes in feed measure viscometric (Sabatier and Fish, 1996).

Measurement of metabolic energy

The balance begin to identify with 18 days (D) as follows:

D 17, birds do not provide forage during the night;

D 18, birds weighed, cleaned Assembly trays;

D 19, feces collect and freeze;

D 20, feces collect and freeze at night do not feed;

D 21, faeces are collected and frozen, the birds are weighed and again fed.

Faeces dried by freezing and crushed as fodder (1 mm, grinder Retsch). The total energy of the feed and excreta was determined by adiabatic colorimeter C5000, IKA. Also determine the protein content (N·6,25, Kjeldahl method Z130) and lipids (method Z160). Adjustment of nitrogen balance type with 18% protein in weight gain.

b. Results and discussion

Obvious exchange energy corrected for nitrogen balance (AMEN)

Zootechnical characteristics and magnitude of the exchange energy is presented in table 1.2. Between treatments no differences in animal performance.

When growing b is Aileron enzyme increases (AME N) feed on the basis of 50% wheat and 22% barley 6.2% (+204 kcal/kg DM (Dry Matter)).

Moreover, the variability of the energy digestibility was reduced from 80 to 62 kcal/kg DM.

The significant increase demonstrates the use of both activities (xylanase and β-glucanase), produced by Penicillum funiculosum against hydrolysis soluble not starchy polysaccharides of wheat and barley.

Table 1.1: Main ingredients and analyzed the characteristics of the experimental feeds
Composition (%)
Wheat50
Barley22
Flour canola8
Bird flour5
Soy flour5
Meat flour5
Fat3
Vitamins/minerals2
Characteristics (%)ControlEnzymes
DM89,789,6
Crude protein20,820,7
Fat54 5,6

Table 1.2.: The influence of the drug enzymes produced by Penicillum funiculosum, on the characteristics of growth and apparent exchange energy for broilers receiving food on the basis of 50% wheat and 22% barley.
Control n=10Enzymes n=10
Total energy intake (kcal/kg DM)46094651
The increase in body mass (g)156±19,8154±22,3
Consumption of food (g/day)104±15,899,7±10,1
FCR (g/g)1,99±0,111,95±0,17
Faeces DM (%)34,7±3,735,6±8,3
AMEN(kcal/kg DM)3252±803456±62
AMEN(kcal/kg DM)2913a±71,93095b±55

Example 2: the Impact of drug enzymes produced by Penicillum funiculosum, on the digestibility of the feed broilers receiving as feed wheat

Carry out tests to determine the effect of the drug enzymes produced by Penicillum funiculosum (activity β-glucanase: 100 u/kg and activity of xylanase: 1100 IU/kg) for obvious submenuoverview (AME), on the digestibility of proteins and lipids broilers receiving feed containing 54% wheat. Explore the dependence of the degree of grinding feed

(1) Control 1 (54% shredded wheat)

(2) Control 1 + Drug enzymes (activity β- glucanase 100 u/kg and activity of xylanase 1100 IU/kg)

(3) Control 2 (30% whole wheat grains, 24% shredded wheat)

(4) Control 2 + Drug enzymes (activity β-glucanase 100 u/kg and activity of xylanase 1100 IU/kg)

In accordance with the European method of comparison (forage ad libitum and complete collection of excreta for age from 18 to 21 days) (Bourdillion et al., 1990).

A. Materials and methods

Birds: the species and growing conditions

Day male Ross broiler chickens kept in cages collective battery until the age of 12 days. Then they are transferred to individual cells to determine the balance digestibility.

Support standard temperature and humidity. Constant light regime is: 23 hours light and 1 hour dark for up to 8 days of age. Then light regime change at 15 h 30 min of light and 8 h 30 min of darkness due to the trials being conducted in the same room.

Feed the Birds get the standard initial feed up to 12 days of age, and then receive the experimental feed.

Experimental feed

Foods contain 54% wheat characteristics to provide the Lena in table 2.1. The feed composition is shown in table 2.2.

Measurement of apparent metabolizable energy

The balance begin to identify with 17 days according to the following scheme:

D 17, birds do not provide forage during the night;

D 18, birds weighed, cleaned Assembly trays;

D 19, feces collect and freeze;

D 20, feces collect and freeze at night do not feed;

D 21, faeces are collected and frozen, the birds are weighed and again fed.

Faeces dried by freezing and crushed as fodder (1 mm, grinder Retsch). The total energy of the feed and excreta was determined by adiabatic colorimeter C5000, IKA. Determine the protein content (N·6,25, Kjeidahl method Z130 for fodder and Z135 for faeces and lipids (method Z160).

The amino acid composition is also determined using HPLC (method Z100 for fodder and Z080 for faeces). The phosphorus content in feed and excreta determine, using the AFNOR method (NFV18-106).

b. Results and discussion

Obvious exchange energy (AME)

Growth characteristics and data about the exchange energy is presented in table 2.3. Characteristics (weight gain, feed consumption), measured over a three-day period not differ between treatments. AME control feed containing 54% shredded wheat is 3173 kcal/kg Metabolic energy feed containing the same quantity of wheat, but 30% of which represent the make whole grain, increased by 100 kcal/kg compared with theoretical value. Moreover, the variability of the estimated standard deviations measured by various criteria, is also reduced when using whole wheat grains.

Enzymes produced by Penicillum funiculosum (activity β-glucanase 100 u/kg and activity of xylanase 1100 IU/kg), increase the value of the exchange energy to feed on the basis of 54% wheat +3,4% (122 kcal/kg DM), if the wheat is crushed, and +2.7% (101 kcal/kg DM), if 30% of wheat included in the form of whole grains.

Apparent digestibility of nutrients (lipids, proteins, and amino acids)

If shredded whole wheat, apparent digestibility of lipids and proteins increased by 7 and 2.7%, respectively, adding the Drug enzymes Penicillum funiculosum. If part of the wheat is presented in the form of whole grains, such an increase is smaller: +3 and +0,6%, respectively, due to the overall increased digestibility of nutrients. Indeed, the absorption of the nutrients for the control feed containing whole grain wheat, similar to the digestibility of the experimental feed containing only shredded wheat, but with the addition of the Drug enzymes.

Effects of enzymes on apparent amino acid digestibility are presented in table 2.4. The improvement when using the Drug enzymes averages +2,9%for shredded wheat and +1.1 per cent for wheat in the form of whole grains, that confirms the effect on the apparent digestibility of protein.

The apparent retention of phosphorus and excretion of phosphorus

Effects of enzymes on the apparent retention of phosphorus are presented in table 2.5. The apparent retention of phosphorus significantly increased when adding the Drug enzymes: +8,0%. This increase is greater than that observed for other nutrients (+2.9 to +3,5% depending on the criteria: AME, proteins, lipids, amino acids). This increase may be the result of increased digestibility of nutrients (the direct effect of xylanase and β-glucanase), but also by the enhanced action of the phytase wheat. As a result of hydrolysis of not starchy polysaccharides xylanase and β-glucanase provide greater accessibility to fitoval acid for endogenous phytase wheat.

This improved the digestibility of phosphorus results in decreased excretion of phosphorus: -8%, if expressed in g phosphorus per kg of weight gain.

Table 2.1. Characteristics of wheat (%)
%Wheat
Dry matter86,2
Crude protein10,87
Lipids1,65
β-glucan0,77
Pentosans6,8
The relative viscositypH 4.5:1,34
(MPa·s)the pH of 1.5:1,29

Table 2.2: Composition and characteristics of the experimental feeds
FoodShredded wheatWhole wheat
Composition (%)
Shredded wheat53,8423,84
Whole wheat030,0
Animal fats3,523,52
Soybean meal 4818,2618,26
Meat and bone meal5,645,64
Peas7,07,0
Whole grains rapeseed10,010,0
Vitamins/minerals1,741,74
Characteristics (%)
ME (kcal/kg)31733,188
Protein20,620,5
Fat9,69,6
Lysine1,05 1,04
Methionine0,450,45
Met+Cys0,850,85
Calcium0,900,90
Available phosphorus0,350,35

Table 2.3: Impact of Drug enzymes (activity β-glucanase: 100 u/kg and activity of xylanase: 1100 IU/kg) on the AMA feed based on wheat (54% shredded wheat or 24% crushed +30% whole wheat grains for growing broilers
Feed1234
Shredded wheatShredded wheat + enzymeWhole wheatWhole wheat + enzyme
The increase in mass (g)172±11,8170±13,1167±8,9165±to 12.0
Feed intake (g)282±20,5272±17,2274±15,5267±14,0
Feed intake per day (g)94±6,891±5,791±5,289±4,7
FCR3(g/g)1,64±0,051,60±0,06 1,64±0,051,63±0,09
Apparent protein digestibility (%)83,8±1,08a85,9±1,14b86,5±0,77bc87,0±0,80c
Apparent digestibility of lipids (%)82,2±2,5a88,0±2,1bc86,6±2,45b89,2±1,25c
AME (kcal/kg DM)3577±76a3699±85b3678±35b3779±34c
(kcal/kg)3194±673303±763284±313375±31
Notes to table 2.3

1: single-way analysis of variance, the effect of the feed, n=47; a, b: values with the same superscripts do not differ at p<0,05.

2: double-way analysis of variance, n=47 (wheat: 54% crushed or 24% crushed +30% whole wheat grains; enzyme; from 0.21/t xylan or without him;

3: FCR: Index of feed conversion (g feed: g weight gain)

Table 2.4: Effects of enzymes on apparent amino acid digestibility (%) of forage on the basis of the 54% of wheat for growing broilers (one mixed sample e is skreta to handle)
Shredded wheatCrushed + whole wheat
TemoinPreparation of enzymesTemoinPreparation of enzymes
Nitrogenof 83.485,386,487,1
ASP78,680,982,182,7
THR74,275,378,079,9
SER79,582,183,083,3
GLU87,989,680,791,4
PRO84,887,187,788,7
GLY77,179,980,782,0
ALA74,676,978,280,1
VAL78,680,881,883,0
ILE80,683,084,085,0
LEU82,184,385,386,4
TYR80,985,083,784,4
PHE83,785,987,187,6
LYS80,683,183,984,8
HIS81,784,985,086,0
ARG84,987,6at 88.189,0
CYS70,872,876,577,0
MET87,288,588,589,4
TRP79,582,783,384,7

Table 2.5: Effects of enzymes on the excretion of phosphorus (P) and apparent retention of phosphorus for food based on wheat (54% shredded wheat) growing broilers (n=12)
Feed12Effect of enzymes
WheatWheat-Drug enzymes
P
The apparent retention of P (%)37,9+3,040,5±2,80,047
Excretion of P (g/bird/3 days)1,24±0,13 1,14±0,10,071
Excretion of P (g/kg prenexa)7,2±0,56,7±0,50,034

Example 3. Evaluation of Drug enzymes in relation N feed based on wheat for growing turkeys

The purpose of this experiment is to demonstrate the efficacy of enzymes from Penicillum funiculosum (activity β-glucanase 100 u/kg and activity of xylanase 1100 IU/kg) with respect to the apparent metabolizable energy (AME) of feed based on wheat in accordance with the following scheme of the experiment

(1) the Control

(2) EP 1: the Preparation of enzymes (activity β-glucanase 100 u/kg and activity of xylanase 1100 IU/kg)

(3) EP 2: Preparation of enzymes (activity β-glucanase 150 u/kg and activity of xylanase 1650 IU/kg)

In accordance with the European method of comparison (Bourdillion et al., 1990): food ad libitum and complete collection of excreta for age from 33 to 37 days.

A. Materials and methods

Birds: the species and growing conditions

Day turkeys BUT9 contain cells with a total battery until the age of 20 days. Then they are transferred to individual cells to determine the balance digestibility after an adaptation period of at least 7 days.

Support standard temperature and humidity. Constant light regime is: 23 hours light and 1 hour of darkness for the first two weeks is, and then photoperiods reduce up to 15 hours of light and 9 hours of darkness until the end of the test.

Feed the Birds receive the standard full initial feed from the first up to 21 days of life, and then receive the experimental feed.

Experimental feed

Foods contain 47% of wheat and 33% soy flour (table 3.1). Spraying enzymes carry 20 kg of particles of the control feed.

Measurement of metabolic energy

On day 21 (D 21) birds are weighed and evenly distributed in 10 separate cells for each treatment, and give experimental feed.

The balance begin to identify with 33 days according to the following scheme:

D 32, birds do not provide forage during the night;

D 33, birds weighed, cleaned Assembly trays;

D-34 and D-35, feces collect and freeze;

D 36, feces collect and freeze at night do not feed;

D 37, faeces are collected and frozen, the birds are weighed and again fed.

Faeces dried by freezing and crushed as fodder (1 mm, grinder Retsch). The total energy of the feed and excreta was determined by adiabatic colorimeter C5000, IKA.

The value of the AME correct nitrogen balance, given the increase in body mass (g) and nitrogen content (21% crude protein).

Faeces dried by freezing and crushed as fodder (1 mm, grinder Retsch). The total energy of the feed and excreta was determined by adiabatic the ski colorimeter C5000, IKA. Determine the protein content (N·6,25, Kjeidahl method Z130 for fodder and Z135 for faeces). The amino acid composition is also determined using HPLC (method Z100 for fodder and Z080 for faeces).

b. Results and discussion the Apparent exchange energy (AME)

Zootechnical characteristics and magnitude of the exchange energy are presented in table 3.2. In the growth characteristics no significant differences in the balance between treatments.

When growing turkeys enzyme increases AMENfeed based on wheat by 2.2% and 5.4% for EP 1 and EP 2, respectively.

So significant the observed increase demonstrates the use of both activities (xylanase and β-glucanase), contained in a Preparation of enzymes against hydrolysis is not starchy polysaccharides of wheat to increase the energy value of this cereal in growing turkeys.

Table 3.1: Main ingredients and analyzed the characteristics of the experimental feeds
Composition (%)
Wheatof 47.55
Extruded soy5,00
Soy flour33,00
Meat flour6,00
Fat4,00
The dicalcium phosphate2,30
Caso30,85
Vitamins/minerals1,30

4680
Characteristics (%)ControlEP 1EP 2
DM89,0of 89.188,9
Crude protein26,326,126,3
Fat6,4
Table 3.2: Effects of enzymes on the obvious exchange energy corrected for nitrogen balance (AMEN) feed based on wheat in growing turkeys (days 32-37). (average ± crotch)
Control n=12EP 1 n=12EP 2 h=12Probability1
Effect of enzymeThe effect of dose
Total energy (kcal/kg DM)46594654
The increase in body mass (g)341±23338±36337,5±57NSNS
Feed intake (g/day)111±5,9107±6,3103±12,1NSNS
The rate of feed conversion (g/g)1,63±0,091,60±0,121,59±0,17NSNS
DM Faeces (%)26,1±5,526,5±2,225,9±4,8NSNS
AMEN(kcal/kg DM)23025±863092±563191±340,0370,061
AMEN(kcal/kg)22700±772753±502840±300,0370,061

1A single way of variance analysis of variance:

Effect of enzymes: n=60; a, b: means that is not marked with the same letters, significantly different at p<0.05; Effect of dose: 0, 0,2, 0,3 I/t.

2Average ± SEM

Example 4. Assessing the impact of Drug enzymes produced by Penicillum funiculosum, on the efficiency of complete feed based on wheat for growing pigs.

The goal is to evaluate the effect of supplementation of enzymes to feed based on wheat on the digestibility of energy in the small intestine of growing pigs. Normal activity levels of the Drug enzymes are 1100 IU/kg of xylanase and 100 IU/kg for β-glucanase.

A. Material and methods

Animals

The control processing is carried out in accordance with the scheme of the Latin square for three feeds and three periods and two pigs for each of the modes of feeding. Forage yield in fixed quantities in accordance with the weight of the pigs during the testing period.

Experimental feed

Feed based on wheat poor quality and balanced at the expense of other typical feed ingredients give six growing pigs (see table 4.1). Give one of the feed:

1. Food without additives (main);

2. Food additives (1): Preparation of enzymes 1 × quantity (activity β-glucanase 100 u/kg and activity of xylanase 1100 IU/kg).

3. Food additives (2): Preparation of enzymes 2 × quantity (activity β-glucanase 200 IU/kg and activity of xylanase 2200 IU/kg).

The exact dose cor is s get diluting the Drug enzymes cornstarch to create a pre-mixture, which is then added to the feed in accordance with the necessity.

Collecting samples

Juices ileum collected within 48 hours each week in accordance with standard procedures in the RPNA laboratories. Energy juice samples of ileum and testing of feed analyzed by calorimetric bomb by Sanders (Sanders) to determine the absorbed energy. Aliquots of the samples stored for further analysis if necessary.

Statistical analysis

The digestibility of crude energy calculated from the results for juice ileum obtained by calorimetric bomb, for fodder and feed intake. Variance analysis undertaken in respect of the calculation of digestibility.

Table 4.1. The composition of ingredients and nutrients main feed
Ingredients% include
Wheat60,0
Barleythe 9.7
Peas11,4
Fish meal5,0
Flour sunflower (30)10,0
Lysine0,15
Minerals and vitamins 3,75
Only100,0
Nutrients
Protein14,9
Dry matter84,9
Digestible energy (kcal/kg)3150
Fiber5,1
Digestible lysine0,8

b. Results and discussion

Supplementation of xylanase to feed pigs increase the digestibility of energy, at least 6%. This indicates that the enzyme enhances the destruction of the cell walls of raw material(particularly wheat) and releases the extra energy in the small intestine

Table 4.2: Impact of the additive Preparation of the enzyme on energy digestibility of feeds based on wheat, which are fed pigs.
Food without additivesAdditive (1)Additive (2)The value p
Average (%)70,174,575,6<0,001
The digestibility of energy0,800,490,45
% improvement6,277,87

Example 5. The influence of St. the ATA enzymes produced by Penicillum funiculosum on the characteristics of fodder from straw, corn silage, hay and grass silage in ruminant animals.

HFT test (Hohenheimer Futterwertesten, Menke et al., 1979, 1988) is an in vitro incubation test to determine the decomposition of the raw material by measuring the volume of gas emitted during fermentation of these forages in buffered juice scar.

A. Material and methods

200 mg of dried and ground substrate incubated with 10 ml juice of scar plus 20 ml of buffer in the syringe, the contents of which were gently mixed on a rotor in an incubator with controlled temperature (39°). Volume of gas evolved register within 24 hours. To amend the results are idle (without substrate), and a standard control with hay and standard control concentrate (with a known quantity of generated gas volume), and expect the full volume of gas produced per 24 hours. Energy value (OMD) (digestible organic matter) substrates calculated using the volume of gas released per 24 hours, and using prediction equations proposed by Menke with TCS. (1988).

Juice scar gather 2 heifers, enter the rumen cannula and fed at 8 a.m. and 7 p.m. food, consisting of 6 kg of hay and 2 kg concentrate (ratio 75/25). Juice scar collected immediately before outran the m feeding. Juice scar filtered, to avoid getting food particles, and contain strictly anaerobic conditions.

The purpose of this test is to verify the influence of additives Preparation of the enzyme for fodder for 15 hours before HFT incubation.

Pre-treatment Preparation of the enzyme: the enzyme solution is sprayed on the feed, spread out on the floor on straw, corn silage, hay and grass silage. The sputtering is carried out, using 1 ml of enzyme for 2 kg of dry matter forage. Fodder from the edges (about 10 cm) is removed to increase the homogeneity of the sample. After processing, the feed is mixed manually and leave at room temperature for 15 hours after spraying. HFT incubation is carried out after 15 hours of contact with the Preparation of enzymes for one series and 6 repetitions for each treatment.

b. Results and discussion

The full volume of gas released per 24 hours, are presented in table 5.1 for straw, corn silage, hay and grass silage.

The use of cellulase for straw in the pre-treatment gives a 18% increase in the full volume of gas compared with the control. For corn silage this increase is 8%, for hay 9.5% and for grass silage 9%. OMD is given in table 5.2 for different types of forage before and after pre-processing.

OM digestibility of the meet is but increases for straw, corn silage, hay and grass silage compared to the control 8.5% for straw, 5% for corn silage, 5.4% for hay and 5% for grass silage.

15 hours pre-treatment of feed (straw, corn silage, hay and grass silage) Drug enzymes increase the intensity of the incubation substrate scar and OM digestibility of the substrate.

Table 5.1: Total volume of gas allocated 24 hours
RAW MATERIALPROCESSINGThe full volume of gas (24 hour)Statistical significance
STRAWControl25S (p<0,05)
Cellulase29,5
CORNControl53,7S (p<0,05)
SILOCellulase57,8
HAYControl39,5(P<0,08)
Cellulaseto 43.1
GRASS SILAGEControl43,7(P<0,08)
Cellulase47,7

Table 5.2: OMD
RAW MATERIALPROCESSINGOMDσStatistical significance
STRAWControl47,00,67S (p<0,05)
Cellulase51,02,37
CORNControl70,70,91S (p<0,05)
SILOCellulase74,22,19
HAYControl59,70,77p<0,08
Cellulase62,9with 3.27
HERBALControl70,50,72p<0,08
SILOCellulase74,0of 3.46

Example 6: Effect of Drug enzymes produced by Penicillum funiculosum on performance of laying hens, the which are fed with wheat or barley

This experiment is carried out to determine the impact of adding the Drug enzymes on the parameters of the productivity rushing chickens fed wheat or barley.

A. Material and methods

Scheme of the experiment: 4 processing × 8 repeats × 5 cells × 3 chicken

Processing: 1. Control 1: 60% wheat

2. Control 1 + medicine enzymes

3. Control 2: 60% barley

4. Control 2 + Drug enzymes

Animals, content and experiment

Experiments conducted on 480 brown chickens strain Nu-Line. Each repetition is carried out for 5 chickens with conventional feeders, i.e. a total of 32 repeat for 15 chickens each.

Distributed in two identical rooms, these repetitions receive programmable lighting and ventilation. The program light is 14 hours of light per day on arrival chickens 17 weeks of age and increased every 2 weeks for 30 minutes up to a maximum of 17 hours of light per day.

Age of hens at the beginning of the experiment 22 weeks, which lasts for 5 months period of egg production.

Feed and feeding

There are two types of experimental feed on the basis of 60% wheat (feed 1) and 60% barley (feed 2), and 10% flour sunflower. Their composition is given in table 6.1. Characteristics of cereals are presented in table 6.2.

Control

Chemical analysis:

The feed samples

Con is the role of the quality of the experimental feeds exercise, analyzing the dry matter, crude protein, crude fat and ash.

The activity of xylanase (T-1, T-2) and the activity β-glucanase (T-3 and T-4) determine in mixed feeds.

Measurement

Feed consumption and feed efficiency recorded every 4 weeks. Chickens are weighed at the beginning and at the end of the experiment. The number of eggs, lots of eggs and % dry matter and fat in eggs recorded daily for 5 periods of 4 weeks each. Mortality in control and record daily, including the cause of death.

b. Results and discussion

The experiment

The obtained parameters of productivity during the experiment are presented in tables 6.3-6.5. In the first two periods (weeks 22-30) throughout the experiment % dirty eggs statistically processed (P>0,005). Birds, which give food without enzymes, are more dirty eggs. Statistical significant difference between treatments was detected for % of eggs laid (P>0.05) and for egg mass (P>0,005), starting from the second period to the end of the experiment. Chickens fed a forage-based barley, show a greater % of eggs laid, and they carried heavier eggs than hens fed wheat. Preparation of enzymes, apparently, increases these parameters, but not significantly, with a value of 0.05 level of probability.

In all experimental periods of consumption to the PMA birds for T-3 and T-4 treatments (feed barley) was higher than the consumption of birds who were given wheat, due to the energy levels of both feed (feed barley corresponds to 2600 kcal/kg energy, whereas wheat forage corresponds to 2800 kcal/kg). Considering the different energy values of feed of both types, and consumption of birds feed during the whole period for all birds daily energy intake was the same.

The feed efficiency (expressed as g feed/g egg) for the experimental feed during the first period was very high because of the small number of eggs for each chicken over this period of time. In the first two periods the efficiency of feed wheat was higher than the efficiency of feed barley; but in the third period, when there was registered a higher percentage of eggs both types of feed presented similar performance. Since 34 weeks before the end of the experiment, feed barley showed higher feed efficiency than wheat forage. Enzymes tended to improve feed efficiency (P>0,05). In all periods β-glucanase increased feed efficiency feed barley (P=of 0.066).

Table 6.4 shows that laying hens fed wheat with the addition of the Drug enzymes tended to demonstrate higher values of egg production (+1.5 abs. points), on average, heavier eggs (+0,37 g) and Bo is its low rate of conversion of feed (-2,7%) compared to the feed without additives.

Table 6.5 shows that the addition of a preparation of enzymes to the barley feed for laying hens increases egg production (+4%) the average mass of eggs (+0.7%) and the rate of conversion of feed (-5,7%) compared with the control feed barley.

Table 6.1: Comparison of experimental feed for hens
IngredientWheat feedBarley feed
Wheat60,171-
Barley-59,033
Animal and vegetable fat (30% linoleic acid)404,0
Extruded soya full fat11,41210,399
Soybean meal, 48%3,4435,705
Flour sunflower, 29%10,010,0
DL-methionine0,0910,101
L-lysine HCl0,111-
Calcium carbonate8,5958,546
The dicalcium phosphate1,4781,517
Sol0,300,30
Preliminary mixture of minerals and vitamins* 0,400,40
Installed nutritional value
The exchange energy (kcal/kg)28002600
Crude protein16,016,41
Crude fatof 7.487,21
Lysine0,750,76
Methionine0,350,35
Methionine + cysteine0,670,69
Calcium3,703,70
Inorganic phosphorus0,400,40
Sodium chloride0,16/a 0.270,15/0,29

(*) 1 kg of feed contains: vitamin A IU ; vitamin D3 1600 IU; vitamin E 5 mg; vitamin K3, 2 mg; vitamin B1 1.5 mg; vitamin B2 4 mg; vitamin B6 3 mg; vitamin B12 to 11.8 µg; folic acid 0.35 mg; Biotin 150 mcg; calcium Pantothenate 10 mg; Niacin 20 mg; Mn 30 mg; Zn, 50 mg; I, 0.3 mg; Fe, 50 mg; Cu, 6 mg; Se, 0.1 mg; ethoxyquin 125 mg

Table 6.2 Analytical composition of cereals
Percentage
Wheat (970556)Barley (970287)
Moisture 10,659,79
Ashof 1.572,11
Crude fat1,761,98
Crude fiber2,43br4.61
Crude protein10,6510,55
Viscosity (pH of 1.5 with inactivation in CP/sec)0,782,31
All β-glucan (%DM)0,663,57
Insoluble β-glucan (%DM)0,501,56
All pentosans4,918,75
Soluble pentosans0,510,39

Table 6.3 Parameters of productivity from 22 to 42 week (full Protocol of the experiment)
ProcessingEgg production %1Dirty eggs (%)Defective eggs (%)Weight of eggs (g)Feed consumption (g)Eff. feed (1)Uves chickens (g)Smart. (%)
T-175,6b7,9a0,862,29b101,5b2,158219,81,7
T-277,1b6,6b 1,162,66b101,5b2,100222,60,8
T-378,0ab5,3b1,063,76a109,4a2,203231,82,5
T-481,2A6,6ab1,064,22a110,0a2,111227,75,0
1,30,40,20,41,50,02816,81,4
The effect of the process.0,04020,00390,77960,00360,00030,06590,95860,2645
P

1Comparison with genetically perfect chickens (data provider chickens). Values are average of 8 repetitions for 15 chickens. Within columns, values with different indexes are significantly different (P<0,05)

Table 6.4: Effect of xylan on the characteristics of hens fed PSH is nice (in absolute values 1and in percent2compared with control).
WeeksThe level of egg production (%)1Weight of eggs (g)1The rate of conversion of feed
12
22-26+1,0-0,3+0,067-1,9%
26-30+1,1+0,7-0,098-4,7
30-34+1,4+0,8-0,039-2,0
34-38+2,1+0,6-0,039-2,0
38-42+2,3-0,5-0,042-2,2
Only+1,5+2,0%+0,37+0,6%-0,058-2,7%

3. For the period 22-42 weeks

Table 6.5: Effects of enzymes on the characteristics of egg production for hens fed the barley (in absolute values1and in percent2compared with control).
WeeksThe level of egg production (%)1Weight of eggs (g)1The rate of conversion of food the
12
22-26+3,7+1,0-0,381-9,0%
26-30+3,0+0,5-0,137-6,3
30-34+1,9+0,5-0,015-0,8
34-38+3,3+0,6-0,067is 3.5
38-42+0,9+0,3-0,082-4,2
Only-3,2+4%+0,46+0,7%-0,092-5,7%

For the period 22-42 weeks

Bibliography

1. Bourdillon, A., Carre, C., Conan L, Duperray J., Franscesch M., Fuentes, M., Huyghebaert G., Jansen W.M.M.A., Leclercq C., Lessire M, J. McNab, It M, Wiseman J., 1990. European reference method for the in vivo determination of metabolizable energy with adult cockerels: reproductibility, effect of age, comparison with predicted values. British Poultry Science 31, 567-576.

2. Sabatier A.M., Fish N.M. 1996. Method of analysis for feed enzymes: methodological problems? Journal of Applied Poultry Research 5, 408-413.

3. Barrier-Guillot Century, Metayer J.P., I. Bouvarel, J. Castaing, Picard M.. Zwick J.L. 1997.

4. Proceedings of the Xlth European Symposium on Poultry Nutrition, WPSA, Aug 24-28 th Faaborg, Denmark, 237-239.

5. Svihus Century, O. Herstad, New man C.W., Newman R.K. 1997. British Poultry Science 38, 524-529.

1. A strain of Penicillium funiculosum IMI378536, producing a complex of enzymes - cellulase, endo-1,4-β-xylanase, cellobiohydrolase, β-glucosidase, and what -1,3(4)-β -glucanase, feruloyl-esterase.

2. The strain according to claim 1, characterized in that it produces at least endo-1,4-β-xylanase and endo-1,3(4)-β-glucanase.

3. The strain according to claim 1, characterized in that it produces at least endo-1,4-β-xylanase, endo-1,3(4)-β-glucanase and cellulase.

4. The strain according to claim 1, characterized in that it produces at least cellobiohydrolase, β-glucosidase, endo-1,3(4)-β-glucanase, feruloyl-esterase.

5. The strain according to claim 1, characterized in that it is used for liquid feed supplements for agricultural animals.

6. The strain according to claim 1, characterized in that it is used for dry feed additives for farm animals.

7. Liquid feed additive for livestock, such as poultry, pigs and ruminants containing microbial product obtained from a strain of Penicillium funiculosum IMI378536 according to claim 1, supplemented, if necessary, the enzyme (enzymes), certain (defined) in claim 1, an antimicrobial agent, sorbitol, antifreeze, filtered concentrated fermentation broth at the following content, wt.%:

Microbial product obtained from
strain Penicillium funiculosum IMI378536
according to claim 1, supplemented, in the case
necessary enzyme (enzymes),
certain (defined) in claim 14-10
Antimicrobial agent0,005-0,35
Sorbitol20-50
Antifreeze0-40
The concentrated filtered
fermentation broth0,3-76

8. The additive according to claim 7, wherein the antimicrobial agent is selected from sorbic acid and its salts, benzoic acid and its salts, methyl-4-hydroxybenzoate, n-propyl-4-hydroxybenzoate, fumaric acid, salts and esters, sodium chloride or potassium chloride.

9. The additive according to claim 7 or 8, characterized in that the antifreeze is chosen from 1,2-propane diol, ethylene glycol, glycerin.

10. The additive according to claim 7, characterized in that it is used to improve the digestibility of cereal crops such as wheat, barley, rye, triticale, oats, rice.

11. The additive according to claim 7, characterized in that it is used to improve the digestibility of oilseeds, such as soybean, sunflower, rape.

12. The additive according to claim 7, characterized in that it is used to improve the digestibility of by-products processing cereals, such as wheat bran

13. The additive according to claim 7, characterized in that it is used to reduce the excretion of phosphorus.

14. The additive according to claim 7, characterized in that it is used to improve the digestibility of phosphorus.

15. The additive according to claim 7, characterized in that it is used to improve the digestibility of amino acids.

16. The additive according to claim 7, characterized in that it is used to reduce the ammonia content in the air battery cell.

17. Dry food for farm animals, such as poultry, pigs and ruminants containing microbial product obtained from a strain of Penicillium funiculosum M according to claim 1, supplemented, if necessary, the enzyme (enzymes), certain (defined) in claim 1, the media, the components of the dry fermentation broth at the following content, wt.%:

Dry microbial product obtained from
strain Penicillium funiculosum IMI378536
according to claim 1, supplemented, in the case
necessary enzyme (enzymes),
certain (defined) in claim 116-40
Media59-83
Components of dry fermentation broth1

18. With the Hoi food for 17, characterized in that the medium is selected from wheat flour, starch, gypsum, maltodextrin, solid products of processing of corn, by-products of the processing of cereals, such as corn grits, wheat forage product, wheat bran, crop roots of rye.

19. Dry food through 17, characterized in that it is used to improve the digestibility of cereal crops such as wheat, barley, rye, triticale, oats, rice.

20. Dry food through 17, characterized in that it is used to improve the digestibility of oilseeds, such as soybean, sunflower, rape.

21. Dry food through 17, characterized in that it is used to improve the digestibility of by-products processing cereals, such as wheat bran.

22. Dry food through 17, characterized in that it is used to reduce the excretion of phosphorus.

23. Dry food through 17, characterized in that it is used to improve the digestibility of phosphorus.

24. Dry food through 17, characterized in that it is used to improve the digestibility of amino acids.

25. Dry food through 17, characterized in that it is used to reduce the ammonia content in the air battery cell.



 

Same patents:

FIELD: microbiology, agriculture.

SUBSTANCE: actinomyces strain Streptomyces lateritius 19/97-M is isolated from tree nursery soil (56°04' North latitude; 92°42 East longitude) in Krasnoyarsk region in 1997 year and deposited in All-Russian Collection of Industrial Microorganissms, FGUPSosNIIgenetics at number VKPM Ac-1637. The actinomyces strain Streptomyces lateritius 19/97-M VKPM Ac-1637 is used for stimulation of growth and protection of coniferous seedlings against pathogens of vascular mycosis. The strain shows high competition ability and growth rate. Invention provides rapid biomass accumulation and high reproductive capacity. Invention can be used for stimulation of growth and protection coniferous seedlings against pathogens of diseases caused by fungi of genus Fusarium and Alternaria.

EFFECT: enhanced effectiveness and valuable properties of strain.

1 tbl, 6 dwg, 3 ex

FIELD: food processing industry, in particular cannery industry.

SUBSTANCE: claimed method includes potato washing, inspection, peeling, post-peeling, cutting, and blanching to obtain half-finished product, pre-packing in vacuum into packets from duplex or laminated film followed by sealing and pasteurization. Prior post-peeling potato is treated with preparation, obtained from biomass of Pythium catenulatum micromycete by sequential extraction with non-polar extractant in above-critical state, water, alkali, water, acid, water, alkali, and water and blending of the first extract with solid precipitate in amount of 0.7-1.105 mg/t and conditioned by approximately 5 hours.

EFFECT: garnish potato of improved organoleptic properties.

FIELD: food processing industry, in particular cannery industry.

SUBSTANCE: claimed method includes potato washing, inspection, peeling, post-peeling, cutting, and blanching to obtain half-finished product, pre-packing in vacuum followed by sealing and pasteurization. Prior post-peeling potato is treated with preparation, obtained from biomass of Mortierella hygrophila micromycete by sequential extraction with non-polar extractant in above-critical state, water, alkali, water, acid, water, alkali, and water and blending of the first extract with solid precipitate in amount of 0.7-1.105 mg/t and conditioned by approximately 5 hours.

EFFECT: garnish potato of improved organoleptic properties.

FIELD: food processing industry, in particular cannery industry.

SUBSTANCE: claimed method includes potato washing, inspection, peeling, post-peeling, cutting, and blanching to obtain half-finished product, pre-packing in vacuum into packets from duplex or laminated film followed by sealing and pasteurization. Prior post-peeling potato is treated with preparation, obtained from biomass of Mortierella minutissima micromycete by sequential extraction with non-polar extractant in above-critical state, water, alkali, water, acid, water, alkali, and water and blending of the first extract with solid precipitate in amount of 0.7-1.105 mg/t and conditioned by approximately 5 hours.

EFFECT: garnish potato of improved organoleptic properties.

FIELD: immunology, proteins.

SUBSTANCE: invention proposes a method for preparing affinity surfaces based on chemically cross-linked staphylococcus protein A. Method involves applying a layer of staphylococcus protein A on hydrophobic surface followed by chemical cross-linkage of protein A. Also, method involves additional applying specific antibodies on the prepared affinity surface with a layer of chemically cross-linked staphylococcus protein A. This method provides preparing the affinity surface eliciting the enhanced strength that can be separated from a backing if necessary and with retaining its integrity. Also, the flat and uniform surface of layer of crossed-linked staphylococcus protein A provides low level of interferences in carrying out study by different methods of microscopy. Invention can be used in investigation of biological objects by different methods, among them by method of atomic-force microscopy. Invention can be used in immunological researches of biological objects.

EFFECT: improved preparing method.

3 dwg, 3 ex

FIELD: veterinary virology.

SUBSTANCE: the virus strain of bursal infectious disease (IBB) "52/70-M" is isolated from bursas of Fabricius of sick and killed chickens. The strain shows the expressed precipitating and infectious activity. Invention can be used for producing inactivated vaccine against IBB virus and for evaluation of properties of vaccines against IBB virus.

EFFECT: valuable properties of strain.

4 tbl, 3 ex

FIELD: biotechnology, microbiology, vitamins.

SUBSTANCE: method relates to a method for preparing riboflavin by culturing the microorganism Bacillus subtilis as a producer in the nutrient medium containing rib-operon from Bacillus amyloliquefaciens, or microorganism able for utilization of glycerophosphate as a single carbon source, or eliciting the resistance against inhibition of growth by glyoxylate, and extraction of riboflavin prepared. Invention uses the following strains as producers of riboflavin: B. subtilis GM51/pMX45, B. subtilis GM41/pMX45, B. subtilis GM44/pMX45. Invention provides preparing riboflavin of the high degree of effectiveness.

EFFECT: improved preparing method.

5 cl, 4 ex

FIELD: biotechnology, microbiology, amino acids.

SUBSTANCE: invention proposes the strain Enterobacter agglomerans/FERM BP-7207 that is able to metabolize the carbon source at pH value when L-glutamic acid precipitates in liquid cultural medium containing L-glutamic acid in the saturation concentration and the carbon source. Also, the strain is able to accumulate L-glutamic acid in the amount exceeding the amount that corresponds to its saturation concentration in liquid cultural medium at this pH value. Also, invention relates to a method for preparing L-glutamic acid by fermentation that involves culturing this microorganism in liquid cultural medium wherein pH value is brought about to the value when L-glutamic acid precipitates to prepare and accumulate L-glutamic acid and to precipitate L-glutamic acid in this cultural medium. The proposed microorganism is prepared by addition of a sample comprising microorganisms to acid cultural medium containing L-glutamic acid in the saturation concentration and the carbon source followed by removal of strain that is able to metabolize this carbon source. Invention provides preparing L-glutamic acid with the high degree of effectiveness.

EFFECT: improved preparing method, valuable properties of strain.

20 cl, 9 dwg, 3 tbl, 8 ex

FIELD: food-processing industry.

SUBSTANCE: method involves preparing and rubbing beet; sterilizing resultant pulp and fermenting while providing combined cultivation thereon of mycelium fungi of Trichoderma kind and of Aspergillus kind of citric acid fermentation; separating cultural liquid and concentrating to dry substance content of 58-60%; introducing resultant concentrate into prepared raw materials and boiling out; introducing into cultural liquid solid residue produced after sequential extracting of Mortierella zychae micromycet biomass with the use of non-polar extractant in above-critical state, water, alkaline, water, acid, water, alkaline, and water, said solid residue being introduced in an amount of 6-8% by weight of dry substances; introducing into concentrate extract produced after extracting of equivalent amounts of the same biomass with the use of non-polar extractant in above critical state.

EFFECT: provision for obtaining of base product with improved organoleptical properties.

5 ex

FIELD: microbiology, agriculture.

SUBSTANCE: invention proposes the strain Pseudomonas aureofaciens IB-6 that exceeds known strains by the level of accumulation of cytokinines. The strain is isolated from sewage waters from aerotank of biological cleaning units and maintained in the Collection of microorganisms of Biology Institute UNTS RAN at number IB-6. The strain in the growth on LB medium forms cytokinines in the amount 680 ng-equiv. of zeatine per 1 ml of medium. Invention can be used for preparing the growth-stimulating preparations used in plant growing by microbiological synthesis.

EFFECT: improved preparing method, high yield of cytokinines.

1 tbl, 2 ex

-glucanase encoding his dna and method thereof" target="_blank">

The invention relates to the field of production of enzyme preparations by means of genetic engineering and can be used in biotechnological processes and microbiological industry

The invention relates to biotechnology and agricultural Microbiology

The invention relates to the field of biotechnology and can be used to produce chitinolytic enzyme preparation

The invention relates to enzyme industry microbiological industry and can be used to obtain highly efficient cellulolyticus enzyme preparation intended for processing vegetable raw materials, obtaining feed and food products from agricultural waste and industrial production

FIELD: genetic engineering, biotechnology, molecular biology, medical-biological and pharmaceutical industry.

SUBSTANCE: invention relates to isolating gene from cells of the strain P. altcromonas producing enzyme that cleaves polysaccharide comprising sulfated fucose and this gene encodes above indicated enzyme, Invention proved the presence of two opened reading frames (ORF-1 and ORF-2) and their nucleotide sequences are determined. Active recombinant forms of enzyme able to hydrolyze sulfated fucose-comprising polysaccharide are obtained by expression of these sequences in E. coli being this polysaccharide is not cleaved by fucoidanase produced by Flavobacterium sp. SA-0082 (FERM BP-5402). Applying the invention provides the possibility for preparing large amounts of qualitative raw for pharmaceutical preparations.

EFFECT: improved preparing method, valuable properties of polypeptide.

5 cl, 5 dwg, 2 tbl, 7 ex

The invention relates to biotechnology can be applied in armoricaine for processing grain portion of the ration
Up!