Strain of filamentous fungus aspergillus oryzae - producer of maltogenic alpha-amylase

FIELD: biotechnology.

SUBSTANCE: strain of fungus Aspergillus oryzae Amy T-52-3-21 produces maltogenic α-amylase, and it is deposited in the All-Russian Collection of Microorganisms Institute of Biochemistry and Physiology of Microorganisms n.a. GK Scriabin RAS under the number F-4476D. The strain is made on the basis of strain Aspergillus oryzae of All-Russian Collection of Microorganisms F-3927D using the genetic engineering methods. Activity of α-amylase at 120 h of growth of the strain is 600-640 units/ml.

EFFECT: higher level of activity of maltogenic α-amylase.

1 tbl, 2 ex


The invention relates to the field of biotechnology and can be used in microbiological industry in the production of acidic α-amylase used in various fields of industry and agriculture for saccharification (hydrolysis) of starch-containing raw materials, namely in baking, distilling, brewing, confectionery and starch industry (production of patok, syrups, glucose), in the production of cereal products, vegetable products, fruit (juices, syrups, extracts, jams, pectin), baby food, textile, paper industry, in the manufacture of detergents, as well as in medicine.

The invention relates to the creation of highly active recombinant strain of the fungus Aspergillus oryzae producer maltogenic α-amylase.

The strain created on the basis of patented industrial producer acidic α-amylase - strain Asp.oryzae VKM F-3927 D, using genetic engineering techniques (1).

The invention provides for obtaining a highly active enzyme preparations of α-amylase in the submerged culture of a new recombinant strain the flour fermentation environments.

α-Amylase (α-1,4-glucan-glucanohydrolase, is an amylolytic enzyme action, belongs to the class of hydrolases, subclass field of glycosidase inhibition, hydrolyzes internal α-1,4-glucose the data communication starch, the glycogen and related poly - and oligosaccharides, resulting in the formation of maltodextrins of various molecular weights, maltotriose, isomaltose and glucose.

Maltogenase α-amylase .oryzae (Taka-amylase) effectively hydrolyzes amylose and amylopectin with the formation of mainly maltose. Role maltogenic α-amylase in the process of bioconversion of starch-containing raw materials currently significantly increased in connection with the development and implementation of technologies for production of fuel ethanol from starch-containing raw materials, involving the use of low-temperature water-heat treatment with the use of highly active acid and enzyme preparations of α-amylase and glucoamylase. Joint use of enzymes helps to increase the speed and depth of the degradation of starch, the degree of hydrolysis of starch reaches 98-99%, while the efficiency of the process of the enzymatic treatment can be greatly enhanced through the use of highly active enzyme preparations.

In the world practice for industrial production of products of hydrolysis of starch is used mainly amylase produced by fungi belonging to the genus Aspergilllus, with yellow-green pigmentation: Asp.flavus Asp.oryzae, Asp.glauscus, Asp.ochraceusi, Asp.fumigatm.

Known for such producers acidic α-amylase, kastama Asp.oryzae 740-A2 (2), Asp.oryzae NRW-169 (collection of the Central Museum of industrial microorganisms "Vniigenetika", collection number CMPM F-257) (3). Also known strains Asp.oryzae 387 (VKPM F-683) (4), Asp.oryzae VKPM F-369 (5), Asp.awamori M-2002 (VKM F-3771D) (6, 7), synthesizing complex hydrolytic enzymes, including acid α-amylase.

The disadvantages of all these producers are the low productivity of the strain applied to the acidic α-amylase. Highly active producers is not available for the organization of domestic production, as are owned by foreign manufacturers.

Closest to the claimed object on the essential features and the achieved result is a strain Asp.oryzae VKM F-3927 D (1)that is under cultivation in deep conditions in flasks for 120 h in culture medium containing as a source of flour of cereals, corn extract, and salt, synthesize α-amylase activity 400-500 IU/ml

However, to increase the profitability of production of enzyme preparations of α-amylase requires the creation of more productive strains.

The problem to which the invention is directed, is getting a new producer strain maltogenic α-amylase capable under cultivation for cheap technological environments to ensure a high level of activity C is left of the enzyme.

The technical result from the creation and use of a new recombinant strain Asp.oryzae ATU T-52-3-21, containing additional copy of the gene maltogenic α-amylase, is to obtain a highly active enzyme preparations sour maltogenic α-amylase for use in various sectors of agriculture.

In order to increase the level of biosynthesis of target enzymes by fungal strains currently successfully applied recombinant DNA technology. Increased expression of secreted enzymes reach by obtaining strains with an increased number of gene copies of the target enzymes (so-called multicopying strains) or directional changes in the mechanism of regulation of the synthesis of these enzymes.

The essence of the object of the invention is a new recombinant strain of Aspergillus oryzae Amy T-52-3-21 - producer maltogenic α-amylase.

The creation of genetically engineered strain based on a mutant strain Asp.oryzae VKM F-3927 D conducted in several stages. In the first stage using induced mutagenesis was obtained auxotrophic recipient strain Aspergillus oryzae 52-3 niaD-defective in nitroreductase, for selection of transformants by their ability to grow on minimal medium with sodium nitrate as the sole nitrogen source. In the second stage, the recipient strain Aspergillus oryzae 52-3 niaD-was cotransformation plasma the Oh frame-Amu, carrying a gene homologous amylase under amylase promoter, plasmid pSTA10, carrier selective marker gene for nitrate reductase (8). In the subsequent breeding was obtained transformant with the level of activity by 20-30% higher than the original strain.

Comparative characterization of the level of activity of α-amylase claimed recombinant strain and initial strain Asp.oryzae VKM F-3927 D. the Activity of α-amylase activity was determined according to GOST 20264.4-89 (9). Table 1 presents data on the biosynthesis of α-amylase original mutant strain (prototype) and recombinant strain Asp.oryzae.

Thus, the proposed strain of Aspergillus oryzae Amu T-52-3-21 under cultivation on a nutrient medium based on wheat and soy flour, provides high activity maltogenic α-amylase activity in the culture fluid, which would greatly increase the yield of the target enzyme with one of the substrate and accordingly reduce the cost of the process of production of enzyme preparations derived based on it.

A strain of Aspergillus oryzae Amu T-52-3-21 deposited in the all-Russian collection of microorganisms at the Institute of biochemistry and physiology of microorganisms them. Gscreen RAS under the number VKM F-4476D.

Culiural-morphological characteristics of the strain Aspergillus oryzae Amu T-52-3-21

Macroscopic characteristics: Colonies on agar of čapek with yeast extract (CA) at 25°C have a diameter of 65-70 mm/7 days greenish-yellow, the surface until fluffy flaky, edge smooth; mycelium dense, white, bleed and have exudate absent, the reverse side unpainted.

Colonies on agar of čapek with yeast extract (CYA) at 37°C have a diameter of 60-65 mm/7 days, olive-yellow, fluffy surface, edge smooth; mycelium dense, white, bleed and have exudate absent, the reverse side unpainted.

Colonies on agar of čapek with yeast extract and 20% sucrose (CY20S) at 25°C have a diameter of colony 67-71 mm/7 days, olive-yellow, fluffy surface to ragged, edge smooth; mycelium dense, white, bleed and have exudate absent, the reverse side unpainted.

Colonies on the best-agar (MEA) at 25°C have a diameter of 65-70 mm/7 days, greenish-yellow, ragged surface, edge smooth; the mycelium is not very dense, white, bleed and have exudate absent, reverse yellowish-gray.

Microscopic characteristics: Conidial heads globose to loosely kolosovidnyh. Conidiophores are not painted, smooth to faintly rough, 500-1000×10-25 μm. Vesicles (apical extension) semi-spherical to pear-shaped, 22-50 µm wide; sterigma single and bunk in one colony, cover 1/2 to 2/3 of the vesicle, Metulla - 8-12×4-6 μm, Fieldy - 8-12×3-5 µm. Conidia globose, about 4.0 to 8.5 microns in diameter, smooth to faintly rough stare at the AI.

Physiological-biochemical properties of strain:

Culture strains metabolizes glucose, sucrose, arabinose, raffinose and poorly - maltose, lactose, galactose and rhamnose. Forms of the enzyme system that allows you to grow at a corresponding complex substrates: starch, β-glucane, pectin and galactomannan.

Well assimilates ammonium salts of inorganic acids. Consumes peptone, casein, amino acids.

Temperature optimum growth 34-35°C, 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, dispensing and delivery of cultures of bacteria, viruses, Rickettsia, fungi, protozoa, Mycoplasma, bacterial toxins, poisons of biological origin".

The obtained recombinant strain differs from the original by the presence in the genome of additional copies of a gene Amu encoding multigene α-amylase (Taka-amylase), which provides an increased ability of a producer to the biosynthesis of α-amylase in submerged cultivation in liquid media and stability transfers.

The strain can be stored in a lyophilized state for several years or jambs with agar medium Saburo or the best agar at 4°C with mandatory replanting at least once in 3 months.

The cultivation of recombinant strains Aspergllus oryzae Amy T-52-3-21 carried out under aerobic conditions at 30°C for 120 h, pH 5.8 and 6.2. For culture growth and biosynthesis of acidic α-amylase source of carbon and nitrogen can serve as starch, hydrolyzed starch, soy, corn or other grain flour, or their extracts, ammonia nitrogen, corn extract.

When the cultivation of the strain Aspergillus oryzae Amy 7-52-3-21 for 120 h in flasks in a medium containing wheat and soy flour, corn extract and inorganic salts, the activity of α-amylase activity in the culture fluid ranges from 580 to 640 IU/ml

The enzyme preparations obtained using the proposed strain, can be used in the form of cultural liquid, concentrated liquid preparations, obtained by ultrafiltration or evaporation of the culture fluid, or in the form of dry preparations (Amiloride GH, GH), obtained by precipitation with ethanol or other organic solvents from ultrafiltrate culture liquid, and drying or granulation.

α-Amylase Asp.oryzae active at a temperature of from 30° to 70°C with optimum action at 58-60°C; stable in wide range of pH values from 3.5 to 8.0, with an optimum activity when pH of 5.0-6.0; more acidic zone, characteristic for flour mixtures (pH 4.5), shows activity at 85-95%; is maltogenic α-amylase, limit the degree of hydrolysis of soluble and insoluble starch is 38 is 34%, respectively.

Table 1
The biosynthesis of α-amylase source and recombinantly strain A.oryzae in the fermentation medium based on wheat flour
No.StrainAC 120 h of growth, IU/ml
1Asp.oryzae VKM F-3927 D (the original strain, prototype)480
2Asp.oryzae Amu T-52-3-21 (recombinant strain)620

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 on liquid nutrient medium composition: wheat flour - 6%, soy flour 4%, corn extract - 1%, NH4NO3to 0.3%, KN2RHO4- a 0.02%, caso31.2%and distilled water, pH 5.8 and 6.2, in flasks containing 50 ml sterile environment, on the rocking chair with 240 rpm at 30°C for 48 hours

Received seed in the amount of 2% by volume of protection is made in Catalogne flask 750 ml containing 50 ml of medium of the following composition (wt.%): wheat flour - 6%, soy flour 4%, corn extract - 1%, NH4NO3to 0.3%, KN2RHO4- a 0.02%, caso3- 1,2%, tap water, pH 5.8 and 6.2. Wednesday pre-treated with the enzyme preparation of bacterial α-amylase at the rate of 2 units/g starch at 70°C for 20 minutes

The cultivation is carried out in aerobic conditions on the rocking chair (240 rpm) at 30°C.

The maximum enzymatic activity of α-amylase (120 h of growth) is 640 IU/ml

Example 2. The cultivation is carried out in katalozhnyh flasks as described in example 1, using liquid nutrient medium of the following composition (wt.%): cornstarch - 10%, soybean flour - 4%, corn extract - 1%, NH4NO3to 0.3%, KN2RHO4- a 0.02%, caso3- 1,2%, tap water, pH 5.8 and 6.2. The maximum enzymatic activity of thermostable α-amylase (120 h of growth) is 600 IU/ml


1. RF patent №2245364 from 27.01.2005.

2. Kalunian K.A., Holger LI Microbial enzyme preparations. Feet, Food industry, 1979, p.8-9.

3. Inventor's certificate SU # 1158579.

4. RF patent №2070921 from 27.12.1996.

5. Inventor's certificate SU # 1440922.

6. RF patent 2196821 from 20.01.2003.

7. Semenov, M.V. and other Microbial biocatalysts for processing agricultural industries/ - M: VNIIBT, 2006. 304 S.

8. Gomi et al. Molecular cloning and characterization of a transcriptional activator gene, amyR, involved n the amylolytic gene expression in Aspergillus oryzae. // Biosci. Biotechnol. Biochem. 2000. V. 64(4). P. 816-827.

9. The enzyme preparations. GOST 20264-4 .89.-M.:Izd. The USSR state Committee for standards, 1995. P.70.

Similar patents of the Russian Federation

1. RF patent №2196821 from 20.01.2003.

2. RF patent №2245364 from 27.01.2005.

3. RF patent №2354697 from 10.05.2009.

The strain of the fungus Aspergillus oryzae Amu T-52-3-21 - producer maltogenic α-amylase, deposited in the all-Russian collection of microorganisms under the number VKM F-4476D.


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