Fungal strain aspergillus oryzae - producer of complex of proteinases and peptidases, nucleases, chitinase, beta-glucanase, mannanase and alpha-amylase

FIELD: biotechnology.

SUBSTANCE: strain Aspergillus oryzae 12-84, having a high level of synthesis of the complex of proteases and peptidases, nucleases, chitinase, β-glucanase, mannanase and α-amylase, is deposited in State Scientific Institution of All-Russian Scientific Research Institute of Agricultural Microbiology of the Russian Agricultural Academy under the registration number Aspergillus oryzae RCAM01134. The strain may be used to produce complex enzyme preparations with their subsequent application for hydrolysis of raw materials in the preparation of biocorrectors of food and feed, amino acid additives and biologically active additives with functional properties.

EFFECT: invention enables to increase the yield of proteolytic, nuclease, chitinase, β-glucanase, mannanase and α-amylase activities.

2 tbl, 2 ex

 

The invention relates to biotechnology and can be used in microbiological, food industry to produce complex products proteinases and peptidases, nucleases, chitinases, β-glucanase, mannanase and α-amylase to further their use for deep hydrolysis of plant, animal and microbial raw materials upon receipt of biocorrectors food and feed, protein and amino acid feed additives and food purposes, biologically active additives with functional properties. The strain of the fungus Aspergillus oryzae 12-84 produces a complex of hydrolytic enzymes and deposited in the collection of useful microorganisms for agricultural purposes wildebeest ARRIAM number Cam system for true 01134.

The invention relates to the establishment of a new mutant strain of the fungus Aspergillus oryzae 12-84 used to obtain the complex of proteinases and peptidases, nucleases, chitinases, β-glucanase, mannanase and α-amylase.

The strain obtained on the basis of known industrial strain of Aspergillus oryzae RUM-156 (VKPM F-981) - producer of proteases by multistep selection with the use of effective methods of mutagenesis.

The invention provides receiving highly active complex of proteolytic enzyme preparations, nucleases, chitinases, β-glucanases and mannanase action in the depth of kultivirovanie� new mutant strain at grain fermentation media.

A distinctive feature of the synthesized enzyme complex is a broad substrate specificity and ability to hydrolyze high molecular weight polymers not only plant and animal raw materials, but also to carry out deep microbial hydrolysis of raw materials, including proteins, nucleic acids and polysaccharides. As a consequence, the strains of microorganisms that produce complex hydrolases, and obtained from it the basis of complex enzymes that catalyze deep hydrolysis of polymers of vegetable, animal, and microbial origin, have broad application prospects in many fields of biotechnology and food industries [1-3]. The use of an enzyme preparation obtained using the proposed strain, allows a deeper hydrolysis of agricultural raw materials, increase the yield and quality of target products, to improve the ecological status of processing industries.

The most typical practical application of the complex preparations of proteolytic, nucleases, chitinases, β-glucanases and mannanase actions related to improving the efficiency of biotechnological processes for the refining of high molecular weight polymers of microbial biomass, including yeast biomass - waste fermentation industries, Miceli� - filled, biomass - waste enzymatic and microbiological industry. The presence in the complex of peptidases, proteinases, nucleases, chitinases, β-glucanase and mannanase allows the use of enzyme preparations for deep microbial hydrolysis of raw materials with the aim of obtaining biologically active food and feed additives with different structural and functional properties.

Known strains of microorganisms - producers of fungal proteases from Aspergillus oryzae 251-90, and 107 387 [4-6], by submerged cultivation used for synthesis of acidic and weakly acidic proteases, the intended and α-amylase. The disadvantages of these producers is the low productivity, narrow complex synthesized enzymes, the absence in the composition of the complex nucleases, chitinases, β-glucanase and mannanase.

Closest to the claimed object is a strain of Aspergillus oryzae RUM-156, registration number VKPM F-981 [7], one of the most active producers of proteases and other hydrolytic enzymes. In-depth culture known strain of Aspergillus oryzae RUM-156 proteolytic activity reaches 28,0-39,0 units PS/cm3, β-glucanase 4,1-4,8 units of β-GCS/cm3.

The disadvantage of this strain is the low level of activity of nucleases, chitinase, β-glucanase and mannanase in the culture fluid (K. g.) of the fungus.

The object of the invention is the obtaining of a strain of the fungus Aspergillus oryzae, the area�ment high ability to form complex proteinases and peptidases, nucleases, chitinases, β-glucanase and mannanase.

The technical result obtained from the use of a new strain of Aspergillus oryzae 12-84, is to obtain a complex enzyme preparation proteinases and peptidases, nucleases, β-glucanase, mannanase, the level of activity which in-depth and solid-phase cultivation exceeds analog: by nucleases in 24-45 time, chitinase - in 3.0-5.0 times, β-glucanase - 1.7-4.5 times, mannanase - 1.4-7.5 times.

The invention is a new mutant strain of Aspergillus oryzae 12-84 - producer of the highly complex hydrolases: proteases and peptidases, nucleases, chitinases, β-glucanase, mannanase and α-amylase.

The problem is solved by the creation of new producer strain complex hydrolases by breeding and mutagenesis from known strain of Aspergillus oryzae POM-156 [7], able to grow on liquid or solid simple in composition of nutrient media and an active synthesis of the complex of proteases, nucleases, chitinases, β-glucanase, mannanase and α-amylase.

As a result of mutagenesis after repeated screening and sieving variants derived strain of Aspergillus oryzae 12-84 - producer of complex proteinases and peptidases, α-amylase with increased biosynthetic capacity against nucleases, chitinases, β-glucanase and mannanase.

Studied the levels of products of the enzymatic complexes of the claimed strain of Aspergillus oryzae 12-84 and famous piece�MMA Aspergillus oryzae RUM-156 when they are deep and solid-phase cultivation by cultivation on solid and liquid nutrient media. General proteolytic activity was determined according to GOST R 53974-2010, using as substrate bovine hemoglobin under the optimal conditions, the actions of proteases, nuclease activity was determined spectrophotometrically by hydrolysis of denatured DNA for 10 min at 40°C, β-glucanase activity - according to GOST R 53973-2010, mannanase activity by the method of Somogi-Nelson, amylolytic activity - according to GOST 54330-2011. Comparative indicators of levels of enzymatic activity in the culture of the fungus Aspergillus oryzae 12-84 with the prototype are presented in table.1 and 2.

Thus, as a result of selection and mutagenesis obtained a new strain of Aspergillus oryzae 12-84 with a high level of synthesis of the complex of proteinases and peptidases, nucleases, chitinases, β-glucanase and mannanase that improves the efficiency of the process of obtaining complex enzyme using Aspergillus oryzae strain 12-84.

The strain is stored as a freeze dried culture and the doorpost with wort-agar in the Department biosynthetic and biocatalytic nanotechnology, enzymes, yeast, organic acids and biologically active substances, State scientific institution all-Russian research Institute of food biotechnology of Russian agricultural Academy, Moscow.

Strain Aspergillus oryzae 12-84 characterized by the following properties.

At 3 days of growth on wort-agar colony size the components�t 37×40 mm, the reverse side is smooth, the profile of the colony is flat, the colony bushy, rounded, smooth edges; the color of the colony - green, margin of colony white fluffy mycelium. On day 6 of growth, the colony diameter 80×82 mm.

During growth on agar medium of čapek size of the colony after 6 days 46×47 mm, color brown colony green, the shape of the colonies are round, smooth edges, white, marked radial folding.

In all environments observed profuse sporulation, the pigments in the environment, not stand out, no exudate, mild smell of mildew.

The morphology of the strain. Vegetative mycelium septate, well-branched with swollen, thick hyphae 5-10 ám; conidia are formed exogenously, the surface of conidia smooth, rounded shape, the diameter of the spores 4-5 ám; conidial forms a head with a single sterigmata. Conidiophores bolivianos form, they are parallel to each other cleopatria of sterigma with conidia.

Physiological and biochemical characteristics. The type of catabolism - breath. Attitude to oxygen aerobic. Optimum growth temperature is 30-32°C, maximum 50°C, minimum 18°C. the Optimum pH environment for fungus growth and biosynthesis of the enzymes of 5.4; the growth of a producer is in the area of pH from 2.5 to 10.0.

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

The non-pathogenic strain.

Strain Aspergillus oryzae 12-84 designed to obtain a complex of proteases, nucleases, chitinase, β-glucanase, mannanase and α-amylase.

By submerged cultivation of Aspergillus oryzae strain 12-84 proteolytic activity is 30.7 units PS/cm3, nucleara is 4.8·10-3units NS/cm3, chitinase - 0,21% cholesterol/cm3, β-glucanase - 6,8% β-GCS/cm3, mandanna - 0,82 units MS/cm3, amylolytic is 12.0 units ░ C/cm3(PL.1).

When growing strain of Aspergillus oryzae 12-84 on wheat bran with moisture content of 54% of the proteolytic activity is 158,7% FS/g nuclease - 36,0·10-3units NS/g, chitinase - 1,74% cholesterol/g, β-glucanases is 46.4% β-GCS/g, mannanase of 12.7 units MS/g, amylase - 123,0 AC units/g (table.2).

The invention is characterized by the following examples.

Example 1. Strain Aspergillus oryzae 12-84 and strain Aspergillus oryzae RUM-156 cultured on a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran - 3,0; KH2PO4To 1.5; the rest is tap water, pH natural. Fermentation nutrient medium inoculated vegetative sowing material of each culture of the fungus in the amount of 4%, grown at 30°C for 18 h. the Deep cultivation of the fungus osushestvliayut Erlenmeyer flasks with a volume of 750 cm 3containing 50 cm3nutrient medium on a pie rocking chair (220-240 rpm) at a temperature of 30-32°C for 48 h. the Maximum level of nuclease, chitinase, β-glucanases and mannanase enzyme activity was observed in the inventive strain of Aspergillus oryzae 12-84 and amounted to: 4,8·10-3units NS/cm3That 0,21% cholesterol/cm3That 6,08% β-GCS/cm3and 0.82 units MS/cm3respectively (table.1).

Example 2. Strain Aspergillus oryzae 12-84 and strain Aspergillus oryzae RUM-156 cultivated on solid nutrient medium of the following composition, %: wheat bran with moisture content of 54%. Fermentation nutrient medium inoculated with an aqueous spore suspension of each culture of the fungus in the amount of 4%. Solid-phase cultivation of the fungus is carried out in conical flasks of 250 cm3containing 50 g of the nutrient medium in stationary conditions at a temperature of 30-32°C for 3 days. The maximum level of proteolytic, nuclease, chitinase, β-glucanases and mannanase enzyme activity was observed in the inventive strain of Aspergillus oryzae 12-84 and amounted to: 158,7 units PS/cm3That 36,0·10-3units NS/cm3That 0,21% cholesterol/cm3, 1,74% β-GCS/cm3and 12.7 units MS/cm3respectively (table.2).

Sources of information

1. Rymarev L. V. Biotechnological aspects of creating food additives bikereviews steps on OS�ove microbial biomass / L. V. Rymarev, E. I. Kurbatova, E. N. Sokolova, N. And., Fursov, V. A. Makarov // Storage and processing of agricultural products. - 2011. - No. 2. - P. 45-47.

2. Rymarev L. V. the Use of biomass of the fungus Aspergillus oryzae as a source of biologically active substances / L. V. Rymarev, E. M. Serb, M. B. Overchenko, K. V. Rachkov, E. V. Orlova, I. M. Abramova // Storage and processing of agricultural products. - 2012. - No. 9. - P. 46-50.

3. Rymarev L. V. Effective proteolytic enzyme preparation and hemicellulases steps for processing agricultural industries/ L. V. Rymarev, M. B. Overchenko, E. M. Serb, K. L. Agaricia // Production of alcohol and liquors. 2010. - No. 4. - P. 14-16.

4. R. V. Zuev Physiological and biochemical studies of experimentally obtained mutant Aspergillus oryzae is an active producer of acid proteinases. Abstract of the Cand. dissertation. - M.: 1971.

5. Patent RF 2070921, CL C12N 1/14, 1993.

6. Patent RF 2315098, CL C12N 1/14, 2006.

7. Patent RF 2315096, CL C12N 1/14, 2006.

The strain of the fungus Aspergillus oryzae RCAM01134 - producer of complex proteinases and peptidases, nucleases, chitinases, β-glucanase, mannanase, α-amylase.



 

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