RU2315095C1 - FUNGUS STRAIN ASPERGILLUS ORYZAE AS PRODUCER OF ACID α-AMYLASE - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-4150 obtained by selection from the known strain Aspergillus oryzae-387 (VKPM F-683) by multistep selection using effective methods of mutagenesis. The strain is stored as lyophilic dried culture and on slants with wort-agar in section of biotechnology of enzyme preparation, in food processing industry department of the State Scientific Institute of VNII food processing technology in Moscow. Invention provides preparing acid α-amylase showing high activity that exceeds activity in analogue by 2-3 times and reducing the process culturing time.

EFFECT: improved and valuable properties of strain.

2 tbl, 5 ex

Description

The invention relates to biotechnology, in particular to the creation of a new strain of Aspergillus oryzae 4150, a producer of acidic α-amylase with high total and specific activity in the culture fluid. The strain was obtained by selection from the known strain Aspergillus oryzae 387 (VKPM F-683) by multi-stage selection using effective methods of mutagenesis. The invention provides a high level of synthesis of acidic α-amylase while reducing the duration of cultivation.

The invention relates to biotechnology, in particular to the creation of a new strain used to obtain the enzyme - acid α-amylase. Amylolytic enzymes are widely used in various industries: alcohol, starch, brewing, wine, baking and others. This is due to the fact that the processed grain raw materials are characterized by a high starch content.

Amylolytic enzymes are obtained as a product of the biosynthetic activity of fungi or bacteria (1, 2). Enzymatic preparations obtained on the basis of microbiological synthesis have different fields of application. So, bacterial producers, for example, Bacillus subtilis 82 (2) synthesize α-amylases having optimum action in the range of pH 6.0-7.5. They are usually used in industries where the process takes place in the neutral pH zone.

Mushroom producers synthesize acidic α-amylases, which exhibit a high level of activity at pH values of 4.5-5.5 and have not only a fluidizing effect, like bacterial α-amylases, but also saccharifying. Therefore, acid α-amylases are widely used in baking and fermentation industries.

In this regard, the selection of new fungal strains to obtain acidic α-amylase is relevant.

A known strain is an active producer of acidic α-amylase from the aspergillus genus, Aspergillus oryzae 740-A-2 (3), during solid-phase cultivation of which acidic α-amylase is synthesized. A disadvantage of this producer is that it synthesizes α-amylase with a high level of activity during surface fermentation.

A known strain of Aspergillus oryzae 3-9-151 (4), with the ability to synthesize α-amylase during deep cultivation with a level of activity in the culture fluid of 3-4 units. AC / cm ³ . However, this strain is characterized by low productivity in deep cultivation.

Closest to the claimed object is a strain of Aspergillus oryzae 387 - one of the most active producers of acidic α-amylase. In the deep culture of the known strains of Aspergillus oryzae 387, amylolytic activity reaches 12-19 units at 48 hours of growth. AC / cm ³ depending on cultivation conditions (5).

The disadvantage of this strain is the low level of amylolytic activity of the culture fluid and the duration of the fermentation.

The objective of the present invention is to obtain a strain of the fungus Aspergillus oryzae, which has a high ability to form acidic amylases, exhibiting maximum activity in the acidic and slightly acidic pH zone.

The technical result obtained from the use of a new strain of Aspergillus oryzae 4150 is to obtain acid α-amylase with high activity in the culture fluid (25-40 units of AC / cm ³ ), which exceeds the analog by 2-3 times while reducing the duration of the cultivation process.

The problem is solved by creating a new strain by selection and mutagenesis from the known strain Aspergillus oryzae 387, capable of rapid growth on simple compositional nutrient media.

The inventive strain Aspergillus oryzae 4150 obtained by multistage selection and mutagenesis using chemical and physical mutagens, in particular ultraviolet and ethyleneimine, deposited in VKPM under No. F-930.

We studied the levels of amylase production in the culture fluid obtained by growing the inventive strain and the known strain in a liquid nutrient medium with aeration and mixing. Concurrent enzymes were acidic proteases and xylanase. Comparative indicators of the levels of amylolytic activity of the culture fluid of the strain Asp.oryzae 4150 with the prototype are presented in table 1. Thus, as a result of selection and mutagenesis, a new strain of Aspergillus oryzae 4150 was obtained, which has a high level of α-amylase synthesis and a shortened cultivation process, which makes it possible to increase the manufacturability of acid amylase enzymes using the strain Aspergillus oryzae 4150. The strain differs from the known strain by cultural and morphological features.

The strain is stored as a lyophilized dried culture on jambs with must-agar in the department of biotechnology of enzyme preparations in the food industry of the state scientific institution of the All-Russian Research Institute of Food Biotechnology in Moscow.

The strain Aspergillus oryzae 4150 is characterized by the following properties.

Cultural signs. After 6 days of growth on the wort agar, colonies with a diameter of 80 × 80 mm are formed, the surface of the colony is fluffy, the colony profile is conical, the colony has a round shape, the edges are wavy, the color is white, the color of the colony turns gray. The color on the back is sand.

When growing on an agarized Chapek medium, the colony size after 6 days is 55 × 55 mm, the color is bright white, the shape of the colonies is round, the edges are wavy.

Weak spore formation is observed on all media, no pigments are released into the medium, no exudate, slight smell of mold.

The morphology of the strain. The fungal mycelium is thin, branched, conidia are formed exogenously; the surface of conidia is smooth, the shape is mainly rounded; thickness hyphae 2-4 microns.

Physiological and biochemical characteristics. The type of catabolism is breathing. Attitude to oxygen - aerob. The optimum growth temperature is 30-32 ° C, the maximum is 50 ° C, the minimum is 18 ° C. The optimal pH value for fungal growth and enzyme biosynthesis is 5.4; producer growth takes place in the pH zone from 2.5 to 10.0.

As a carbon source, the fungus uses starch, glucose, sucrose, xylose, maltose, mannitol, glycerin, galactose. The producer assimilates nitrates, ammonium and amine nitrogen, proteins.

The strain is non-pathogenic.

The strain Aspergillus oryzae 4150 was used to produce acidic α-amylase.

The invention is characterized by the following examples.

Example 1. The strain Aspergillus oryzae 4150 is cultivated on a nutrient medium of the following composition,%: barley flour - 3.0; wheat bran - 3.0; KH ₂ PO ₄ - 1.5; the rest is tap water, the pH is natural. The fermentation medium is inoculated with vegetative seed in an amount of 4%, grown at 30 ° C for 18 hours. The cultivation of the fungus is carried out in Erlenmeyer flasks with a volume of 750 cm ³ containing 50 cm ³ on a circular rocking chair (220-240 rpm) at a temperature of 30-32 ° C for 42 hours. The activity of extracellular α-amylase is 33.6 units. AC / cm ³ .

Example 2. The strain Aspergillus oryzae 4150 is cultured on a nutrient medium of the following composition,%: starch - 2.0; barley flour - 3.0; wheat bran - 3.0; KN ₂ RO ₄ - 1.5; the rest is tap water (medium 1), pH is natural. Inoculation of the nutrient medium and the cultivation of the strain are carried out analogously to example 1. The activity of extracellular α-amylase is 25.0 units. AC / cm ³ .

Example 3. The strain Aspergillus oryzae 4150 is cultivated on a nutrient medium of the following composition,%: wheat flour - 6.0; wheat bran - 1.0; KH ₂ PO ₄ - 1.5; the rest is tap water (medium 2), pH is natural. Inoculation of the nutrient medium and the cultivation of the strain are carried out analogously to example 1. The activity of extracellular α-amylase is 36.5 units. AC / cm ³ _.

Example 4. The strain Aspergillus oryzae 4150 is cultivated on a nutrient medium of the following composition,%: wheat flour - 6.0; wheat bran - 2.0; KH ₂ PO ₄ - 1.5; the rest is tap water (medium 3), the pH is natural. Inoculation of the nutrient medium and the cultivation of the strain are carried out analogously to example 1. The activity of extracellular α-amylase is 37.2 units. AC / cm ³ .

Example 5. The strain Aspergillus oryzae 4150 is cultured on a nutrient medium of the following composition,%: wheat flour - 6.0; wheat bran - 2.0; K ₂ HPO ₄ - 1.5; the rest is tap water (medium 4), pH is natural. Inoculation of the nutrient medium and the cultivation of the strain are carried out analogously to example 1. The activity of extracellular α-amylase is 40.0 units. AC / cm ³ .

Table 1 Comparative evaluation of the physiological activity of Aspergillus oryzae fungi, producers of acidic α-amylase Strain Duration of cultivation, hour Amylolytic activity, units AC / cm ³ 387 48 12.0 387 120 19.0 107 42 33.6 table 2 The influence of culture media on the enzymatic activity of Aspergillus oryzae 4150 fungus No.
nutrient medium Enzymatic activity, units / cm ³ AC PS The cop one 25.0 4.8 1,2 2 36.5 2.1 4.2 3 37,2 1.3 2,5 four 40,0 1.9 0.9

Information sources

1. Gracheva I.M. Technology of enzyme preparations. - M .: Food industry. 1975.

2. A.S. USSR No. 1084297, cl. C12N 1 / 20.1984.

3. Kislukhina OV Enzymes in the production of food and feed. - M .: DeLi print. 2002.

4. Dvadtsatova E.A. Obtaining active amylolytic enzymes from deep Aspergillus cultures. - M .: CINTI food industry. 1961. Part 1.

5. RF patent 2070921, cl. 12 N 1/14, 1993.

Claims (1)

The strain of the fungus Aspergillus oryzae 4150, VKPM F-930 - producer of acidic α-amylase.