RU2315096C1 - FUNGUS STRAIN ASPERGILLUS ORYZAE AS PRODUCER OF COMPLEX OF PEPTIDASES, β-GLUCANASE, α-AMYLASE AND XYLANASE - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae ROM-156 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 preparations in food processing department of the State Scientific Institute VNII food processing technology in Moscow. Invention provides preparing the complex of proteinases and peptidases wherein their level of activity in cultural fluid exceeds activity of analogue by 6-10-fold, 1.3-1.4-fold and 1.6-1.9-fold with respect to β-glucanase, α-amylase and xylanase, respectively. The process cultivation time of the strain is reduced by 1.6-1.9-fold.

EFFECT: improved and valuable properties of strain.

3 tbl, 4 ex

Description

The invention relates to biotechnology, in particular to the creation of a new strain of Aspergillus oryzae POM-156 - a producer of a complex of proteinases and peptidases, α-amylase, xylanase, β-glucanase; optimum action of pH 4.5-5.5 with high productivity of exohydrolases. The strain was obtained by selection from the known strain of Aspergillus oryzae 387 (VKPM F-683) by multi-stage selection using effective mutagenesis methods. The invention provides a high level of synthesis of a complex of proteolytic, amylolytic and cellulolytic enzymes of hemicellulase action with an in-depth method of culturing a producer.

The invention relates to biotechnology, in particular to the creation of a new strain used to obtain a complex of proteinases and peptidases, α-amylase, xylanase and β-glucanase. Proteolytic enzymes are widely used in various industries: alcohol, meat and dairy, brewing, winemaking, agricultural and others. This is due to the fact that the processed agricultural raw materials contain high molecular weight protein polymers. The most typical practical use of complex preparations of proteolytic, amylolytic and cellulolytic effects is associated with the intensification of biotechnological processes for the production of alcohol, beer, etc. The presence of peptidases, proteinases, and β-glucanase in the complex allows the use of enzyme preparations for proteolysis of microbial protein in order to obtain biologically active food and feed additives. Hydrolytic enzymes are obtained as a product of the biosynthetic activity of fungi or bacteria [1, 2]. However, the use of fungi is more preferable than bacteria, because they synthesize a complex complex of proteases, which include active peptidases and proteinases, as well as a rich complex of concomitant enzymes that more efficiently catalyze high molecular weight microbial, plant and animal feed polymers in the acidic and weakly acidic pH zones . In this regard, the selection of new fungal strains to obtain a complex of hydrolases is relevant.

Known strains are producers of fungal proteases from the genus Aspergillus oryzae 251-90, 824-32 [3, 4], during the deep cultivation of which acidic and weakly acidic proteases are synthesized. The disadvantages of these producers are their low productivity, the complex composition of the nutrient media, as well as the long period of growth of the producers.

Closest to the claimed object is the strain Aspergillus oryzae 387, registration number VKPM F-683 [2], one of the most active producers of proteases and a complex of other hydrolytic enzymes. In the deep culture of the known strain of Aspergillus oryzae 387 for 42 hours of growth, the proteolytic activity reaches 11.5 units. PS / cm ³ depending on the composition of the nutrient medium.

The disadvantage of this strain is the low level of protease activity and concomitant enzymes in the culture fluid (cf) of the fungus.

The objective of the present invention is to obtain a strain of the fungus Aspergillus oryzae, which has a high ability to form a complex: proteases containing active carboxy and aminopeptidases and proteinases, as well as concomitant enzymes - α-amylase, β-glucanase and xylanase.

The technical result obtained from the use of a new strain of Aspergillus oryzae ROM-156 is to obtain a more active complex of proteinases and peptidases, the activity level of which is 2.5-3.9 times higher than the analogue, as well as concomitant enzymes with high activity in the culture fluid, exceeding the analogue: for β-glucanase - 6-10 times, for α-amylase - 1.3-1.4 times, for xylanase - 1.6-1.9.

The problem is solved by creating a new strain - a producer of the hydrolase complex by selection and mutagenesis from the known strain Aspergillus oryzae 387 (5), capable of rapid growth on simple composition nutrient media and the active synthesis of a complex of proteases, β-glucanase, α-amylase and xylanase.

The inventive strain Aspergillus oryzae POM-156 obtained by multistage selection and mutagenesis using effective chemical and physical mutagens, deposited in VKPM under No. F-931.

As a result of mutagenesis, after repeated selection and screening of variants, the strain Aspergillus oryzae POM-156 was obtained - a producer of a complex of proteases, α-amylase, β-glucanase and xylanase, which has a high growth rate and productivity of the complex of these enzymes.

We studied the production levels of the enzymatic complex in the culture fluid obtained by growing the inventive strain and the known strain in a liquid nutrient medium with aeration and mixing. The total proteolytic activity was determined by the modified Anson method using bovine hemoglobin as a substrate under optimal protease conditions, amylolytic activity according to GOST 20264.4-89, xylanase according to Shomody-Nelson, β-glucanase by glucose oxidase method. Comparative indicators of the levels of enzymatic activities of the culture fluid of the strain Aspergillus oryzae POM-756 with the prototype are presented in table 1 and table 3.

Thus, as a result of selection and mutagenesis, a new strain of Aspergillus oryzae ROM-156 was obtained, which has a high level of synthesis of a complex of proteases: peptidases and proteinases, β-glucanase, as well as α-amylase and xylanase and a reduced duration of the cultivation process, which allows to increase the processability of the process for obtaining a complex of enzymes using the strain Aspergillus oryzae POM-156. The strain differs from the known and cultural-morphological characteristics.

The strain is stored in the form of freeze-dried culture and 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 POM-156 is characterized by the following properties.

Cultural signs. After 6 days of growth on the wort agar, colonies with a diameter of 86-88 mm are formed, the surface of the colony is rough, folded, the profile of the colony is flat, the shape of the colony is round, the edges are wavy, the color is light green, the color of the colony darkens with age. The color on the back is sand.

When growing on an agarized Chapek medium, the colony size after 6 days is 45-47 mm, the colony color is brown-green, the shape of the colonies is round, the edges are even, white, radial folding is noted.

Abundant spore formation is observed on all media, pigments are not released into the medium, there is no exudate, and a slight smell of mold.

The morphology of the strain. The fungal mycelium is septate, well branched, with swellings, conidia are formed exogenously; the surface of conidia is smooth, the shape is mainly rounded; the thickness of the hyphae is 4-8 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 POM-156 was used to obtain a complex of proteases, β-glucanases, as well as amylolytic and xylanolytic enzymes.

Under deep cultivation of the strain Aspergillus oryzae ROM-156, depending on the conditions, the proteolytic activity is 28.0-39.2 units. PS / cm ³ , amylolytic - 14-17 units. AC / cm ³ , β-glucanase - 3.8-4.8 units. β-GCS / cm ³ , xylanase - 5.6-7.8 units. KS / cm ³ (table 3).

The invention is characterized by the following examples.

Example 1. The strain Aspergillus oryzae POM-156 and strain 387 are cultured on a nutrient medium of the following composition,%: barley flour - 3.0; wheat bran - 3.0; KN ₂ RO ₄ - 1.5; the rest is tap water, the pH is natural (medium 1). 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 of} fermentation medium on a circular shaker (220-240 rpm) at a temperature of 30-32 ° C for 38 hours. The maximum accumulation of enzymes was observed in the strain A.oryzae ROM-156 by 38 hours of cultivation. The level of activity was: for protease - 28.0 units. PS / cm ³ for β-glucanase - 4.1 units β-GCS / cm ³ , for α-amylase - 14.0 units. AC / cm ³ for xylanase - 6.8 units. KS / cm ³ (table 1). The synthesized complex of proteases includes: serine proteinase - 0.081 units / cm ³ , carboxylic acid - 1.19 units / cm ³ , metal-dependent - 0.412 units / cm ³ , leucine aminopeptidase - 2.3 units / cm ³ , carboxypeptidase - 0.79 units / cm ³ (table 2).

Example 2. The strain Aspergillus oryzae POM-156 is cultivated on a nutrient medium of the following composition,%: barley flour - 3.0; wheat bran - 3.0; corn columns - 1.0 KH ₂ PO ₄ - 1.5; the rest is tap water, the pH is natural (medium 2). Fermentation conditions similar to example 1. The activity of extracellular proteases is 39.2 units. PS / cm ³ , β-glucanase - 4.8 β-GCS / cm ³ , amylolytic - 17.0 units. AC / cm ³ , xylanolytic - 7.8 units. KS / cm ³ .

Example 3. The strain Aspergillus oryzae POM-156 is cultivated on a nutrient medium of the following composition,%: barley flour - 4.0; soybeans - 1.5; KH ₂ PO ₄ - 1.5; the rest is tap water, the pH is natural (medium 3). Fermentation conditions similar to example 1. The activity of extracellular proteases is 32.2 units. PS / cm ³ , β-glucanase - 4.2 β-GCS / cm ³ , amylolytic - 14.3 units. AC / cm ³ , xylanolytic - 5.5 units. KS / cm ³ .

Example 4. The strain Aspergillus oryzae POM-156 is cultured on a nutrient medium of the following composition,%: barley flour - 4.0; soybeans - 0.5; malt sprouts - 1.0; KH ₂ PO ₄ - 1.5; the rest is tap water, pH is natural (medium 4). Fermentation conditions similar to example 1. The activity of extracellular proteases is 31.4 units. PS / cm ³ , β-glucanase - 4.3 β-GCS / cm ³ , amylolytic - 14.0 units. AC / cm ³ , xylanolytic - 5.7 units. KS / cm ³ .

Table 1 Comparative evaluation of the physiological activity of Aspergillus oryzae fungi - producers of proteases Strain Duration of cultivation, hour Proteolytic activity, units PS / cm ³ PS β-GCS AC The cop 387 42 11.5 0.4 10.5 4.1 ROM-156 38 28.0 4.1 14.0 6.8

table 2 Biochemical composition of enzymatic complexes synthesized by Aspergillus oryzae micromycete Name of the enzyme Strain A.oryzae 387 POM-56 Total proteolytic activity of RN-5.5, units PS / cm ³ 11.5 28.0 Proteinases Serine 0,047 0,081 Carboxylic 0.603 1,190 Metal dependent 0.195 0.412 Peptidases Leucine 0.98 2,30 Carboxy 0.37 0.79

Table 3 The influence of culture media on the enzymatic activity of the fungus Aspergillus oryzae pcs. 387 and pcs. ROM-156 Strain Nutrient medium Enzymatic activity PS β-GCS AC The cop units / cm ³ % increase units / cm ³ % increase units / cm ³ % increase units / cm ³ % increase 387 one
2
3
four 9.7
10.1
9.8
9,4 -
-
-
- 0.5
0.8
0.6
0.7 -
-
-
- 11.3
12.3
10.8
10,2 -
-
-
- 3.8
4.2
3.4
3.2 -
-
-
- ROM-156 one
2
3
four 30,0
39.2
32,2
31,4 210
288
228
234 3.8
4.8
4.2
4.3 660
500
600
515 15.0
17.0
14.3
14.0 32
38
32
37 6.2
7.8
5.5
5.7 63
86
62
78

Information sources

1. Gracheva IM, Krivova A.YU. Technology of enzyme preparations. - M.: Publishing. The Elevator, 2000.

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

3. Zueva R.V. Physiological and biochemical studies of the experimentally obtained mutant Aspergillus oryzae, an active producer of acidic proteinase. Abstract of Cand. dissertations. - M., 1971.

4. A.S. USSR No. 1440922, class C12N 1/14, 1988.

5. RF patent 2070921, 1993.

Claims (1)

The strain of the fungus Aspergillus oryzae ROM-156, VKPM F-931 is a producer of a complex of proteinases and peptidases, β-glucanase, α-amylase and xylanase.