RU2514224C1 - Strain of filamentous fungus aspergillus oryzae - producer of maltogenic alpha-amylase - Google Patents

Abstract

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.

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Description

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

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

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

The invention provides for the production of highly active enzyme preparations of α-amylase as a result of deep cultivation of a new recombinant strain on flour fermentation media.

α-Amylase (α-1,4-glucan-glucanohydrolase, 3.2.1.1) is an amylolytic action enzyme, belongs to the class of hydrolases, a subclass of glycosidases, hydrolyzes internal α-1,4-glucosidic bonds in starch, glycogen and related poly- and oligosaccharides, leading to the formation of maltodextrins of various molecular weights, maltotriose, isomaltose and glucose.

Maltogenic α-amylase A.oryzae (Taka-amylase) effectively hydrolyzes amylose and amylopectin to form mainly maltose. The role of maltogenic α-amylase in the bioconversion of starch-containing raw materials has now increased significantly in connection with the development and implementation of a technology for the production of fuel ethanol from starch-containing raw materials, which involves the use of low-temperature water-heat treatment using highly active and acid-resistant enzyme preparations of α-amylase and The joint use of enzymes contributes to an increase in the speed and depth of degradation of starch; as a result, the degree of hydrolysis of starch reaches 98-99%, while the efficiency of the enzymatic treatment process can be significantly increased due to the use of highly active enzyme preparations.

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

Producers of acidic α-amylase are known, such as strains Asp.oryzae 740-A2 (2), Asp.oryzae НРВ-169 (collection of the Central Museum of Industrial Microorganisms "VNIIGenetika", collection number TsMPM F-257) (3). Also known are strains of Asp.oryzae 387 (VKPM F-683) (4), Asp.oryzae VKPM F-369 (5), Asp.awamori M-2002 (VKM F-3771D) (6, 7), synthesizing a complex of hydrolytic enzymes including acidic α-amylase.

The disadvantages of all of the above producers are the low productivity of the strains in relation to acidic α-amylase. Highly active producers are not available for the organization of domestic production, since they are the property of foreign manufacturing companies.

The closest to the claimed object by the set of essential features and the achieved result is the strain Asp.oryzae VKM F-3927 D (1), which, when cultivated in deep conditions in flasks for 120 hours on a nutrient medium containing cereal flour as a source, corn extract and salts, synthesizes α-amylase with an activity of 400-500 u / ml.

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

The problem to which the invention is directed, is to obtain a new producer strain of maltogenic α-amylase capable of providing a high level of activity of the target enzyme when cultured on low-cost technological media.

The technical result obtained from the creation and use of a new recombinant strain Asp.oryzae Atu T-52-3-21, containing an additional copy of the gene for maltogenic α-amylase, is to obtain highly active enzyme preparations of acidic maltogenic α-amylase for use in various branches of the agro-industrial complex.

To increase the level of biosynthesis of target enzymes by fungal strains, recombinant DNA technologies are currently successfully used. Increases in the expression of secreted enzymes are achieved by obtaining strains with an increased number of copies of the genes of the target enzymes (the so-called multicopy strains) or by a directed change in the regulation mechanism 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 of maltogenic α-amylase.

The creation of a recombinant strain based on the mutant strain Asp.oryzae VKM F-3927 D was carried out in several stages. At the first stage, using the induced mutagenesis, the auxotrophic recipient strain Aspergillus oryzae 52-3 niaD ^- , defective in nitrate reductase, was obtained to select transformants for their ability to grow on minimal medium with sodium nitrate as the sole source of nitrogen. At the second stage, the recipient strain Aspergillus oryzae 52-3 niaD ^{- was} co ^- transformed with the plasmid pAMu-Amu carrying the homologous amylase gene under the amylase promoter, with plasmid pSTA10 carrying the selectable marker nitrate reductase gene (8). As a result of subsequent selection, a transformant with an activity level of 20-30% higher than the initial strain was obtained.

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

Thus, the proposed strain Aspergillus oryzae Amu T-52-3-21 when cultivated on a nutrient medium based on wheat and soy flour, provides a high activity of maltogenic α-amylase in the culture fluid, which can significantly increase the yield of the target enzyme per unit substrate and, accordingly, reduce the cost of the production process of enzyme preparations obtained on its basis.

The strain Aspergillus oryzae Amu T-52-3-21 was deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms named after G.K.Skryabin RAS under the number VKM F-4476D.

Culipural-morphological features of the strain Aspergillus oryzae Amu T-52-3-21

Macroscopic characteristics: Colonies on апapek agar with yeast extract (CYA) at 25 ° C have a diameter of 65-70 mm / 7 days, greenish-yellow, the surface is fluffy to flocculent, the edge is even; the mycelium is dense, white, exudate is absent, the reverse side is unpainted.

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

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

Colonies on Maltz Agar (MEA) at 25 ° C have a diameter of 65-70 mm / 7 days, greenish-yellow, the surface is ragged, the edge is even; the mycelium is not very dense, white, there is no exudate, the reverse side is yellowish-gray.

Microscopic characteristics: Conidial heads are spherical to loose columnar. Conidiophores are not colored, smooth-walled to slightly rough, 500-1000 × 10-25 microns. Vesicles (apical extensions) hemispherical to pear-shaped, 22-50 microns wide; sterigms are single-tier and two-tier within the same colony, cover from 1/2 to 2/3 of the vesicle surface, metula - 8-12 × 4-6 microns, phialids - 8-12 × 3-5 microns. Conidia are spherical, 4.0-8.5 microns in diameter, smooth-walled to slightly rough during aging.

Physiological and biochemical properties of the strain:

The strain culture assimilates glucose, sucrose, arabinose, raffinose and weakly maltose, lactose, galactose and rhamnose. It forms enzyme systems that allow it to grow on the corresponding complex substrates: starch, β-glucan, pectin and galactomannan.

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

The temperature optimum of growth is 34-35 ° C, pH 4.5-6.0. Aerobe.

This type of mycelial fungus is not listed as pathogenic in the "Regulation on the registration, storage, handling, dispensing and transfer of cultures of bacteria, viruses, rickettsia, fungi, protozoa, mycoplasmas, bacterial toxins, poisons of biological origin."

The resulting recombinant strain differs from the initial one by the presence in the genome of an additional copy of the Amu gene encoding maltogenic α-amylase (Taka-amylase), which provides increased producer ability to α-amylase biosynthesis during deep cultivation in liquid media and stability during transfers.

The strain can be stored in a lyophilized state for several years or on jambs with agar medium Saburo or Malz-agar at + 4 ° C with mandatory reseeding at least once every 3 months.

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

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

Enzyme preparations obtained using the proposed strain can be used in the form of culture fluid, concentrated liquid preparations obtained by ultrafiltration or evaporation of the culture fluid, or in the form of dry preparations (Amilorizin G10x, G20x) obtained by precipitation with ethanol or other organic solvents from ultrafiltrate of the culture fluid, as well as drying or granulation.

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

Table 1 Biosynthesis of α-amylase with the original and recombinant A.oryzae strain on a wheat flour-based fermentation medium No. Strain AS for 120 h of growth, units / ml one Asp.oryzae VKM F-3927 D (initial strain, prototype) 480 2 Asp.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 essence of the claims associated with them.

Example 1. Sowing material (inoculum) is obtained by growing a strain on a liquid nutrient medium composition: wheat flour - 6%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0.3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, distilled water, pH 5.8-6.2, in flasks containing 50 ml of sterile medium, on a shaker with 240 rpm at 30 ° C for 48 h .

The resulting seed in an amount of 2% by volume of the medium is introduced into rocking flasks with a volume of 750 ml containing 50 ml of medium of the following composition (wt.%): Wheat flour - 6%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0.3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, tap water, pH 5.8-6.2. The medium is pretreated with an enzymatic preparation of bacterial α-amylase at the rate of 2 units / g of starch at 70 ° C for 20 minutes.

Cultivation is carried out under aerobic conditions on a rocking chair (240 rpm) at a temperature of 30 ° C.

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

Example 2. Cultivation is carried out in rocking flasks, as described in example 1, using a liquid nutrient medium of the following composition (wt.%): Corn starch - 10%, soy flour - 4%, corn extract - 1%, NH ₄ NO ₃ - 0.3%, KH ₂ PO ₄ - 0.02%, CaCO ₃ - 1.2%, tap water, pH 5.8-6.2. The maximum enzymatic activity of thermostable α-amylase (120 h of growth) is 600 units / ml.

Sources of literature

1. RF patent №2245364 from 01/27/2005.

2. Kalunyants K.A. and Golger L.I. Microbial enzyme preparations. M., Food Industry, 1979, p. 8-9.

3. Copyright certificate SU No. 1158579.

4. RF patent No. 2070921 dated 12/27/1996.

5. Copyright certificate SU No. 1440922.

6. RF patent 2196821 from 01.20.2003.

7. Semenova M.V. et al. Microbial biocatalysts for the processing branches of the agro-industrial complex / - M.: VNIIIPBT, 2006. 304 p.

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

9. Enzyme preparations. GOST 20264-4.89.-M.: Publ. USSR State Committee for Standards, 1995. P.70.

Similar patents of the Russian Federation

1. RF patent No. 2196821 from 01.20.2003.

2. RF patent №2245364 from 01/27/2005.

3. RF patent No. 2354697 dated 05/10/2009.

Claims (1)

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