Fungus strain aspergillus oryzae as producer of acid α-amylase

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

 

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

The invention relates to biotechnology, in particular to the creation of a new strain used to obtain enzyme - sour α-amylase. Amylolytic enzymes are widely used in various industries: alcohol, starch, brewing, winemaking, baking, and other. This is because processed grain raw material is characterized by a high content of starch.

Amylolytic enzymes get as the product of the biosynthetic activity of fungi or bacteria (1, 2). The enzyme preparations obtained on the basis of microbiological synthesis, have different applications. Thus, bacterial producers, for example, Bacillus subtilis 82 (2) synthesize α-amylase having the optimum action in the field of pH values of 6.0 and 7.5. They usually are used in industries where the process takes place in Nate the social area pH.

Mushroom producers synthesize sour α-amylase having a high level of activity at pH values of 4.5-5.5 and has not only thinning effect, as bacterial α-amylase, but saharauis. So sour α-amylase is widely used in baking and brewing industries.

In this regard, the selection of new fungal strains to get sour α-amylase relevant.

A known strain - active producer of sour α-amylase from the kind of Aspergillus - Aspergillus oryzae 740-A-2 (3), for solid-phase cultivation which is synthesized sour α-amylase. The disadvantage of this producer is that it synthesizes α-amylase with a high level of activity in surface fermentation.

A known strain of Aspergillus oryzae 3-9-151 (4), with the ability to synthesis α-amylase in submerged cultivation with the level of activity in the culture fluid 3-4 units ° C/cm3. However, this strain is characterized by low productivity in submerged cultivation.

Closest to the claimed object is a strain of Aspergillus oryzae 387 is one of the most active producers of sour α-amylase. Deep culture known strains of Aspergillus oryzae 387 48 hours growth amylolytic activity reaches 12-19 units ° C/cm3depending on the culture conditions (5).

Under taccom this strain is a low level of amylase activity in the culture fluid and the duration of fermentation.

The problem posed by the present invention is to obtain a strain of the fungus Aspergillus oryzae, which has high ability to generate acid amylase exhibiting maximum activity in the acidic and weakly acidic zone pH.

The technical result from the use of a new strain of Aspergillus oryzae 4150 is getting sour α-amylase with high activity in the culture fluid (25-40 units ° C/cm3exceeding the equivalent in 2-3 times while reducing the time consuming process of cultivation.

The problem is solved by the creation of new strains through breeding and mutagenesis of a known strain of Aspergillus oryzae 387, capable of rapid growth on simple nutrient media.

The inventive strain of Aspergillus oryzae 4150 obtained by multistage breeding and mutagenesis using chemical and physical mutagens, in particular UV and ethylenimine deposited in ACIM # F-930.

Studied the levels of production of amylase in the culture fluid obtained by growing the claimed strain and known strain in a liquid nutrient medium with aeration and stirring. Related enzymes when this was acidic protease and xylanase. Comparative indicators of levels of amylase activity in the culture fluid of strain Asp.oryzae 4150 with the prototype presented in table 1. Therefore clicks the zoom, as a result of breeding and mutagenesis received a new strain of Aspergillus oryzae 4150, high-level synthesis α-amylase and the shortened duration of the cultivation process, which improves the manufacturability of the process of obtaining acidic enzymes amylases when using a strain of Aspergillus oryzae 4150. Strain differs from known and cultural-morphological characters.

The strain is stored in a lyophilized culture on the shoals with wort-agar in the Department of biotechnology enzyme preparations in the food industry of the state scientific institution all-Russian research Institute of food biotechnology, Moscow.

A strain of Aspergillus oryzae 4150 is characterized by the following properties.

Cultural characteristics. After 6 days of growth on wort-agar formed colonies with a diameter of 80×80 mm, the surface of the colony fluffy, profile colonies conical form colonies rounded, edges wavy, color - white, with age, the color of the colony of grey. The color on the reverse side of the sand.

During growth on agar medium of čapek size colonies after 6 days 55×55 mm, color bright white, form colonies rounded, edges wavy.

In all environments, there is weak sporulation, the pigments in the environment are not highlighted, exudate no, weak odour of mildew.

Morphology of strain. The fungal mycelium thin, branched, conidia are formed aksogan is about; the surface of conidia smooth, shape, mostly rounded; height hyphae 2-4 ám.

Physiological and biochemical characteristics. Type catabolism - breath. Relation to oxygen - aerobe. Optimum growth temperature is 30-32°maximum - 50°C, the minimum is 18°C. the Optimal pH environment for fungus growth and biosynthesis enzymes 5,4; the growth of a producer takes place in the zone of pH from 2.5 to 10.0.

As a source of carbon to the fungus uses starch, glucose, sucrose, xylose, maltose, mannitol, glycerol, galactose. Producer assimilates nitrate, ammonium and amine nitrogen, proteins.

The non-pathogenic strain.

A strain of Aspergillus oryzae 4150 used to get sour α-amylase.

The invention is characterized by the following examples.

Example 1. A strain of Aspergillus oryzae 4150 cultivated in a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran to 3.0; SC2PO4to 1.5; the rest is water, pH natural. Fermentation nutrient medium inoculated vegetative seeds in the amount of 4%, grown at 30°C for 18 hours. The fungus cultivation is carried out in Erlenmeyer flasks with a volume of 750 cm3containing 50 cm3on the pie rocking chair (220-240 rpm) at a temperature of 30-32°C for 42 hours. The activity of extracellular α-amylase is 33.6% AU/cm .

Example 2. A strain of Aspergillus oryzae 4150 cultivated in a nutrient medium of the following composition, %: starch - 2,0; barley flour - 3,0; wheat bran to 3.0; SC2RHO4to 1.5; the rest is water (environment 1), pH - natural. Sowing medium and cultivation of the strain are as in example 1. The activity of extracellular α-amylase was 25.0% AU/cm3.

Example 3. A strain of Aspergillus oryzae 4150 cultivated in a nutrient medium of the following composition, %: wheat flour - 6,0; wheat bran - 1,0; KN2PO4to 1.5; the rest is water (medium 2), pH natural. Sowing medium and cultivation of the strain are as in example 1. The activity of extracellular α-amylase 36.5% AU/cm3.

Example 4. A strain of Aspergillus oryzae 4150 cultivated in a nutrient medium of the following composition, %: wheat flour - 6,0; wheat bran - 2,0; KN2PO4to 1.5; the rest is water (medium 3), pH natural. Sowing medium and cultivation of the strain are as in example 1. The activity of extracellular α-amylase is 37,2% AU/cm3.

Example 5. A strain of Aspergillus oryzae 4150 cultivated in a nutrient medium of the following composition, %: wheat flour - 6,0; wheat bran - 2,0;2HPO4to 1.5; the rest is water (environment 4), pH natural. For the EB culture medium and the cultivation of the strain are as in example 1. The activity of extracellular α-amylase is 40,0% AU/cm3.

Table 1
Comparative evaluation of physiological activity from Aspergillus oryzae - producers sour α-amylase
StrainDuration of cultivation, hAmylase activity, units ° C/cm3
38748to 12.0
38712019,0
1074233,6
Table 2
Effect of nutrient media on the enzymatic activity of the fungus Aspergillus oryzae 4150
No.

nutrient medium
Enzymatic activity, units/cm3
AUPSCA
125,04,81,2
236,52,14,2
37,21,32,5
440,01,90,9

Sources of information

1. Grachev IM Technology of enzyme preparations. - M.: the Food industry here. 1975.

2. As the USSR №1084297, CL 12N 1/20,1984.

3. Kalugina O.V. Enzymes in the production of food and fodder. - M.: Delhi print.

4. Dvadcatoe E.A. Getting active amylolytic enzymes from deep cultures of Aspergillus. - M.: CINTI the food industry. 1961. Part 1.

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

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



 

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