Fungus strain aspergillus oryzae as producer of acid and subacid proteases

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to development of a novel strain used for preparing enzyme representing a complex of acid subacid proteases. Strain is prepared by selection from the known strain Aspergillus oryzae (VKPM F-683) by multistep selection using effective methods of mutagenesis. Strain is stored as lyophilic dried culture and on slants with wort-agar in the biotechnology section of enzyme preparations in the food processing department of the State Scientific Institute VNII of food processing technology in Moscow. Invention provides preparing the strain possessing the high level of synthesis of acid and subacid proteases and high total activity of enzyme in cultural fluid exceeding activity of analogue by 2.0-2.6-fold and in reducing culturing process 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 107 - producer acidic proteases 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 of acidic proteases, thereby reducing the duration of cultivation.

The invention relates to biotechnology, in particular to the creation of a new strain used to obtain enzyme - complex acidic and weakly acidic proteases. Proteolytic enzymes are widely used in various industries: alcohol, meat, dairy, brewing, wine, agriculture and others. This is because processed agricultural raw materials contains high molecular weight protein polymers. The most typical practical application of complex products, acidic and weakly acidic proteases due to the intensification of biotechnological processes in the fermentation industries for production of alcohol, beer, meat and dairy industry, cheese making, etc. Acidic proteases are also used for microbial proteolysis of the protein with the aim of obtaining biologically active food and feed additives. Proteolytic shall armenti receive as a product of the biosynthetic activity of fungi or bacteria (1, 2). However, the use of fungi rather than bacteria because they synthesize complex of proteases, which includes active peptidases and proteases, implementing more efficient catalysis of protein polymers in the acid zone pH. In this regard, the selection of new fungal strains to obtain acidic proteases relevant.

Known strains - producers of fungal proteases of the genus Aspergillus oryzae 251-90, 824-32 (3, 4), with deep cultivation which are synthesized acidic and weakly acidic protease. The disadvantage of these producers is their low productivity, complex nutrient media, as well as the long period of growth producers.

Closest to the claimed object is a strain of Aspergillus oryzae 387, registration number VKPM F-683 (2), one of the most active producers of proteases. Deep culture known strain Asp. Oryzae 387 42 hours growth of proteolytic activity reaches of 11.5 units PS/cm3depending on the composition of the nutrient medium.

The disadvantage of this strain is the low level of proteolytic activity in the culture fluid (k.j), especially in acidic pH (2,5-4,0).

The problem posed by the present invention is to obtain a strain of the fungus Aspergillus oryzae, which has high ability to form complex proteases exhibiting the maximum is aktivnosti in acidic and weakly acidic zone pH.

The technical result from the use of a new strain of Aspergillus oryzae 107 is getting acidic and weakly acidic proteases with high total activity in the culture fluid, exceeding similar in 2.0-2.6 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 (5), capable of rapid growth on simple nutrient media.

The inventive strain of Aspergillus oryzae 107 obtained by multi-stage selection using effective methods of mutagenesis, deposited in ACIM # F-929.

As a result of mutagenesis after repeated selection and screening of alternatives was obtained a strain of Aspergillus oryzae 107 producing acidic and weakly acidic proteases with high growth rate and productivity of complex acidic and weakly acidic proteases.

Studied the production of acid and weakly acidic proteases in the culture fluid obtained by growing the claimed strain and known strains in liquid nutrient medium with aeration and stirring. General proteolytic activity was determined by the modified method of the memory using as substrate bovine hemoglobin at different values of pH and optimum temperature of action of proteases. Comparative figures ur the init proteolytic activity of the culture liquid strain Asp.oryzae 107 with the prototypes presented in table 1.

Thus, through selection and mutagenesis received a new strain of Aspergillus oryzae 107 high-level synthesis of proteases and the shortened duration of the cultivation process, which improves the manufacturability of the process of obtaining enzymes acidic and weakly acidic proteases using strain Aspergillus oryzae 107. Strain differs from known and cultural-morphological characters.

The strain is stored in a lyophilized culture and the doorpost with wort-agar, 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 107 is characterized by the following properties.

Cultural characteristics. After 6 days of growth on wort-agar formed colonies with a diameter 50-53 mm, surface rough colonies with concentric circles, profile colonies flat, form colonies irregular, wavy edge, color - green, the edges are light yellow with age colony color darkens. The color on the reverse side of gray.

During growth on agar medium of čapek size colonies after 6 days 30-32 mm, color colonies of brown-green, the colonies are round, smooth edge.

In all environments, there is abundant sporulation, the pigments in the environment are not highlighted, exudate no, the faint smell of mold

Morphology of strain. The fungal mycelium septate, branched, with swellings, conidia are formed exogenous; the surface of conidia smooth, shape, mostly rounded; height hyphae 4-6 microns.

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 107 used to get acidic and weakly acidic proteases.

The invention is characterized by the following examples.

Example 1. A strain of Aspergillus oryzae 107 cultured on 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 (20-240 rpm) at a temperature of 30-32° With over 38 hours. The activity of extracellular proteases is: at pH 2,5 - 3,6 units PS/cm3; at pH 4.0 - 12,6 units PS/cm3; at pH 4.5 - 18,0 units PS/cm3; at pH 5.5 to 18.5 units PS/cm3.

Example No. 2. A strain of Aspergillus oryzae 107 cultured on a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran to 3.0; SC2RHO4to 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 28°C for 40 hours. The activity of extracellular proteases is 19.9 units PS/cm3.

Example No. 3. A strain of Aspergillus oryzae 107 cultured on a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran to 3.0; SC2RHO4to 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°C for 40 hours. The activity of extracellular Protea what is 20,8% PS/cm3.

Example No. 4. A strain of Aspergillus oryzae 107 cultured on a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran to 3.0; SC2RHO4to 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 32°C for 40 hours. The activity of extracellular proteases is 21.5 units PS/cm3.

Example No. 5. A strain of Aspergillus oryzae 107 cultured on a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran to 3.0; SC2RHO4to 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 34°C for 40 hours. The activity of extracellular proteases is 20.5 units PS/cm3.

Table 1
Comparative assessment is and physiological activity from Aspergillus oryzae - producers of proteases
StrainDuration of cultivation, hProteolytic activity, units PS/cm3
pH 2,5pH 4.0pH 4.5pH 5.5
251-90720,6a 3.9the 5.75,9
387422,06,911,011,5
107384,118,023,022,5
Table 2
The effect of temperature growth on the proteolytic activity of the fungus Aspergillus oryzae St and St
StrainThe duration of growth, hProteolytic activity, units PS/cm3
28°30°32°34° 36°
38740

48
8,9

10,5
10,1

11,9
8,5

9,2
6,0

5,3
4,2

3,1
10740

48
19,9

21,0
20,8

21,3
24,8

23,9
20,5

19,5
17,5

to 12.0

References

1. Grachev I.M., A. Krivova Technology of enzyme preparations. - M.: Publishing House. "Elevator". 2000.

2. RF patent 2208633, C 12 N 1/20, 2001.

3. Zueva W. Physiological and biochemical studies of experimentally obtained mutant Aspergillus oryzae is an active producer of acid proteases. Abstract of Cand. the dissertation. - M.: 1971.

4. As the USSR №1440922, CL 12N 1/14, 1988.

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

The strain of the fungus Aspergillus oryzae 107, VKPM F-929 - producer acidic and weakly acidic proteases.



 

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