The strain of filamentous fungus aspergillus awamori - producer of glucoamylase
(57) Abstract:The invention relates to biotechnology, can be used for saccharification of starchy raw materials in various food industries that require highly active enzyme preparations that are resistant to acidic pH values. A strain of Aspergillus awamori N 400 VKM F-D 3689 received multistage genetic breeding of the original strain of Aspergillus awamori VKM F-758 with the use of ultraviolet irradiation with simultaneous exposure to chemical mutagens. The strain has a high glucoamylase activity. Glucoamylase active in the pH range of 3.5-7.5 and a temperature of 30-65oC. To obtain a high productivity of the strain can be used nutrient medium, traditionally used in industrial technologies for obtaining this kind of enzyme preparations that do not require complex methods of cultivation of the producer. The invention relates to the microbiological industry, and various areas of food biotechnology and can be used to obtain glucoamylase with the purpose of its further use for the enzymatic treatment of starch-containing raw material in the alcohol, starch, baking and brewing industry.
Macroscopic characteristics: colonies on agar of čapek with yeast extract (CYA), 25oWith, have a diameter 70-71 mm/cut, radially furrowed, velvety surface, edge thin, conidial region dark brown; bleed and have exudate absent, reverse dull yellow.Colonies on agar of čapek with yeast extract (CYA), 37oWith, have a diameter 70-71 mm/sut., cultural characteristics similar to those on CYA, 25oC.Colonies on agar of čapek with yeast extract and 20% sucrose (CY20S), 25oWith, have a diameter of colony 68 - 70 mm/sut., weak radial-grooved, surface porosity, edge thin, conidial region dark brown; bleed and have exudate absent, reverse dull yellow.Microscopic characteristics: conidial heads globose, then richardella decaying into separate columns, conidiophores slightly coloured in terminalname over the entire surface. Sterigma mostly bunk, Metulla 6-16 x 3.5 -7 μm, Fieldy 5-8 x 2 - 4 microns. Conidia globose, 3.5 to 6 μm, smooth.Physiological and biochemical properties of the strain
Relation to sugars
Culture strains metabolizes glucose, sucrose, arabinose, raffinose, and poorly - maltose, lactose, galactose and rhamnose.Starch hydrolyses to glucose.Well assimilates ammonium salts of inorganic acids. Consumes peptone, casein, amino acids. Peptonized milk.Temperature optimum growth 34-35oWith a pH of 4.5-6.0. Aerobe.This filamentous fungus is not listed as pathogenic in the "Regulations on the procedure for accounting, storage, handling, issuance and delivery of cultures of bacteria, viruses, Rickettsia, fungi, protozoa, Mycoplasma, bacterial toxins, poisons of biological origin".The resulting mutant in their morphological properties different from the original intensity of sporulation, the color pigment dispute, the color of the colony on the reverse side when grown on agar media, a shorter growth cycle, the formation of the more abundant biomass, increased ability to biosynthesis of glucoamylase with globin is of 3-6% atypical colonies, that indicates a high morphological stability of the strain, and therefore, its stability in the products of glucoamylase during prolonged cultivation.Cultivation of the strain is carried out in aerobic conditions at a temperature of 35oWith over 120-144 h, pH of 5.2 to 5.5. For culture growth and biosynthesis of glucoamylase source of carbon and nitrogen can serve as starch, hydrolyzed starch, flour extrudate cereals (corn, wheat, barley, rye), ammonia nitrogen, corn extract.The strain has a high potential for the synthesis of glucoamylase, and also produces minor amounts of amylase, protease and glucanase.For enzymatic treatment of starch-containing raw material in the production of alcohol enzyme preparation can be used in the form of cultural liquid or in the form of concentrated products known biochemical methods, such as precipitation with ethanol from ultrafiltrate cultural liquid.Glucoamylase active in a wide range of pH from 3.5 to 7.5 with an optimum at pH 4.5 to 5.0; in the temperature range from 30 to 65oWith optimum at 60 - 62oC. Glucoamylase stable for 2P CLASS="ptx2">The possibility of using the invention is illustrated by examples which do not limit the scope and nature of the claims associated with them.Example 1. Seed material (AMF inoculum) is produced by cultivation of a strain of Aspergillus awamori BKM F-3689 D on solid nutrient medium consisting of 50% wheat bran and 50% malt sprouts, moistened with tap water up to 60%, pH of 5.2 to 5.5. Vyrashivayu spore inoculum is carried out in flasks containing 20-30 g of a solid substrate, in stationary conditions at a temperature of 35oC for 5-6 days and when 22-23oC for 7 days.Received seed in the amount of 0.08 to 0.1% by volume environment contribute in Catalogne flask 750 ml containing 100 ml of medium composition, g/l: corn flour - 240,0; tap water - the rest, pH 5,2-5,5.Cultivation of the strain is carried out in aerobic conditions at a temperature of 35oOn the rocking chair with 240 rpm for 120-144 hours Every 24 hours, starting from 72 h of growth, take samples, which determine the capacity of the culture fluid to hydrolyze starch.Method for determining the activity of glucoamylase based on the determination of glucose formed by hydrolysis of soluble starch is acting on soluble starch at 30oC and a pH of 4.7 for 1 min, liberates 1 µmol of glucose.Maximum glucoamylase activity in the culture fluid at 120 h of growth is 400 IU/ml at 144 h of growth - 450 u/ml Specific glucoamylase activity in the culture fluid equal to 15.5 IU/mg protein.Example 2. The method is carried out as in example 1, but in a fermentation medium instead of corn flour used extrudate (corn flour) in the amount of 250 g/lMaximum glucoamylase activity in the culture fluid at 144 h of growth 500 u/mlExample 3. The method is carried out as in example 1, but the fermentation medium instead of corn flour contains wheat flour in the amount of 180 g/L.Maximum glucoamylase activity in the culture fluid at 144 h of growth 380 IU/mlThus, the proposed strain Aspergillus awamori BKM F - 3689 D can significantly improve glucoamylase activity in the culture fluid and, therefore, increase the efficiency of enzyme preparations on the basis of this strain in various fields of biotechnology.In addition, to obtain high production strain does not require the use of complex nutrient media and complex p is used in industrial production technologies such enzyme preparations.LITERATURE
1. U.S. patent N 4335208, CL 195-5, publ. 19822. Wongwicham A et. al.: Biotechnol. Bioeng, Nov 20; 65(4), 1999.3. Withersy M et. al.: Biotechnol. Bioeng., 1998, Aug 20; 59(4), 407-418.4. Pederson et. al. : Appl. Environ. Biotechnol., 2000, Mar 53(3), 272-276.5. Bocring Sp.: Biotechnol. Bioeng, 1999, Dec 20; 65(6), 638-648.6. USSR author's certificate N 800185, CL 12 D 13/10, publ. 19817. USSR author's certificate N 1094346, CL 12 N 9/34, publ. 19858. USSR author's certificate N 1271068, CL 12 N 9/34, publ. 19949. GOST 20264-4.89. The strain of filamentous fungus Aspergillus awamori BKMF-D 3689 (all-Russian collection of microorganisms at ibfm them. G. K. Skryabin RAS) - producer of glucoamylase.
FIELD: biotechnology, biochemistry.
SUBSTANCE: the strain Aspergillus awamori VKM F-3771 D is obtained by the multiple-stage genetic selection of the parent strain Aspergillus awamori VUD T-2 F-203 using ultraviolet irradiation. The strain elicits high activity of enzyme glucoamylase. For providing the high productivity of the strain nutrient media can be used that are used usually in industrial technologies in preparing similar enzyme preparations being without using complex procedures in culturing the strain-producer. Invention can be used for saccharification of starchy raw in different branches of food industry wherein highly active enzyme preparations with resistance to acid pH values are required.
EFFECT: valuable properties of strain.
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FIELD: biotechnology, microbiology.
SUBSTANCE: invention relates to producing ethyl alcohol or fodder for monogastral animals. The polyenzyme product with glucoamylase, proteolytic and xylanase activities is prepared by fermentation of wheat bran using microorganism Aspergillus niger. Indicated glucoamylase, proteolytic and xylanase activities have the following minimal values: glucoamylase activity - at least 100 U/g of dry matter; proteolytic activity - at least 100 U/g of dry matter; xylanase activity - at least 100 U/g of dry matter under condition that glucoamylase activity has to be at least 750 U/g of dry matter and/or xylanase activity has to be at least 300 U/g of dry matter. Invention provides enhancing the soluble nitrogen content in wort after saccharification, reducing viscosity and effectiveness in using.
EFFECT: improved preparing method, valuable properties of hydrolyzed bran.
14 cl, 10 tbl, 7 ex
SUBSTANCE: method provides the hydrolysis of starch-containing raw materials, following adding of the mineral salts to the obtained hydrolysate and medium fermentation with acid-forming fungi Aspergillus niger. The hydrolysis is carried out with ferment preparate of bacterial α-amilase at increased temperature and excessive pressure. The starch-containing raw material are rice flour allowed beforehand in the water at 20-25°C during 10-12 hrs. in amount of 205.3-235.7 g and rye flour in amount of 232.0-290.6 g. The carbon and nitrogen ratio in the obtained rice flour suspension is provided respectively equal (20.0-23.0):1, in rye flour suspension - (14.0-16.0):1. The ferment preparate is added up to achieving of carbon source dextrose equivalent value 35-40%. The sugars conversion to citric acid is 86.7-93.2%. α-amilase and glucoamylase activity is respectively 4.5-6.2 units A,C/cm3 and 160-180 units Gl.C/cm3.
EFFECT: increase of α-amilase and glucoamylase yield while maintaining the high yield of citric acid.
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SUBSTANCE: invention is a DNA molecule encoding an enzyme having glucoamylase activity, possessing at least 80% sequence identity with glucoamylase from Trichoderma, possessing the sequence of SEQ ID NO: 4. The invention also refers to vectors and host cells with integrated DNA sequences, enzyme compositions and methods of use of glucoamylase in various applications.
EFFECT: expansion of arsenal of glucoamylases.
45 cl, 20 dwg, 10 tbl, 8 ex
SUBSTANCE: invention relates to versions of glucoamylase with altered properties and to methods of glucoamylase versions application.
EFFECT: reduced formation of condensation products and increase of glucose output during starch conversion.
22 cl, 2 tbl, 1 ex
SUBSTANCE: strain is prepared of a commercial producer, a mutant strain, Aspergillus awamori M-2002 (Russian National Collection of Microorganisms F-3771D) with using methods of induced mutagenesis and DNA technologies. The strain A.awamori Xyl T-15 is deposited in Russian National Collection of Microorganisms of Scryabin Institute of Biochemistry and Physiology of Microorgnisms of Microorgnisms of the Russian Academy of Sciences No. 4278D, stored in a lyophilised condition on a mowed wort agar in the Department of Enzymatic Preparation in Food Industry of State Scientific Institution All-Russian Research Institution of Food Biotechnology of Moscow Russian Agricultural Academy.
EFFECT: invention provides producing high-active complex enzymatic preparations of glucoamylase and xylanase with high activity of xylanase with respect to an initial strain to be applied in various fields of agricultural sector.
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SUBSTANCE: strain is prepared of a commercial producer, a mutant strain, Aspergillus awamori M-2002 (Russian National Collection of Microorganisms F-3771D) with using methods of induced mutagenesis and plasmid transformation. The strain A.awamori amyR - T-19 is deposited in Russian National Collection of Microorganisms of Scryabin Institute of Biochemistry and Physiology of Microorgnisms of the Russian Academy of Sciences No. 4277D, stored in a lyophilised condition on a mowed wort agar in the Department of Enzymatic Preparation in Food Industry of State Scientific Institution All-Russian Research Institution of Food Biotechnology of Moscow Russian Agricultural Academy.
EFFECT: invention provides considerably higher glucoamylase yield from a substratum unit.
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SUBSTANCE: group of inventions relates to feed production, particularly a method for microbiological production of feed yeast from grain wastes. The method involves preparation of grain material by grinding to 120-160 mcm particles. Further, the ground grain material is used to prepare an aqueous suspension containing 15-25% dry substances. The obtained suspension is saccharified with α-amylase and glucoamylase to 6-10% glucose content in the suspension. Nitrogen and phosphorus sources are added to the obtained suspension such that 90 mg of nitrogen and 45 mg of phosphorus are required for synthesis of 1 g of biomass. A culture is added to the obtained nutrient medium, said culture being a producer of the Saccharomyces cerevisiae yeast strain VKPM U-3585, having amylase activity, which is deposited in the Russian National Collection of Industrial Microorganisms (VKPM) and can be used in producing feed protein. The Saccharomyces cerevisiae yeast strain VKPM U-3585 is continuously grown in a multiple-section apparatus while feeding the nutrient medium simultaneously into several sections, followed by concentration of the suspension by vacuum evaporation and drying. Moisture freed at the vacuum evaporation and drying steps is used to prepare the aqueous suspension of grain material.
EFFECT: invention increases output of crude protein and true protein.
SUBSTANCE: invention relates to biotechnology and can be used in production of forage protein products. Method includes preparation of grain raw material (bran, substandard grain, fodder flour) by its milling to particles with size 1-20 mcm by means of mill under pressure with shift and preparation of water suspension of grain raw material with concentration of dry substances 15-16% with ratio of grain raw material and water 1:5. Carrying out fermentolysis of grain raw material by means of liquid α-amylase and glucoamylase with formation of glucose to 10-13%. Introduction of nitrogen and phosphorus sources into obtained fermentolysat; as nitrogen and phosphorus sources used are ammonium or phosphate salts or fertilisers, including ammophos, diammophos, nitrophosphate, with obtaining nutritional medium. Introduction into nutritional medium of culture-producent, as such used is strain of yeasts Saccharomyces cerevisiae Y-3585, and continuous growing in multisectional growing apparatus with supply of nutritional medium simultaneously by several sections with further concentration of suspension on vacuum-evaporator and drying. Moisture, released at stages of vacuum-evaporation and drying, is re-used for preparation of water suspension of grain raw material.
EFFECT: invention makes it possible to increase content of raw protein in finished product and simplify method of production of forage protein product.
5 cl, 7 tbl, 9 ex
FIELD: food industry.
SUBSTANCE: invention is related to a method of preparing beer wort. Method envisages addition in the glucoamylase mash, that is at least 90% identical to sequence, shown in SEQ ID NO: 1, the mash contains malted and non-malted grains.
EFFECT: use of the said glucoamylase for mashing allows using smaller amount of enzyme with reduced time of mashing and improved profile of sugar in the wort.
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