RU2315097C1 - Fungus strain aspergillus oryzae as producer of acid proteases and xylanase - Google Patents

Abstract

FIELD: biotechnology, microbiology, biochemistry.

SUBSTANCE: invention relates to the strain Aspergillus oryzae-12 providing the high level of synthesis of acid proteases and xylanase. The strain is obtained by multistep selection of the strain Aspergillus oryzae-387 (VKPM F-683) using effective methods of mutagenesis. The strain is deposited in collection VKPM at № F-932. Invention provides preparing the enzyme complex showing the high proteolytic and xylanolytic activity in cultural fluid wherein their level exceeds activity in analogue by 2.5-2.8-fold.

EFFECT: valuable properties of strain.

2 tbl, 3 ex

Description

The invention relates to biotechnology, namely, to obtain a new strain of Aspergillus oryzae 12 - producer of a complex of acidic proteases and xylanases with high total activity in the culture fluid. The strain was obtained 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 a complex of acidic proteases and xylanase.

The invention relates to biotechnology, in particular to the creation of a new strain used to obtain a complex of enzymes, acidic and weakly acidic proteases, xylanase. Proteolytic and xylanolytic enzymes are widely used in various industries: alcohol, brewing, winemaking, feed and others. This is due to the fact that the processed agricultural raw materials contain high molecular weight protein polymers and plant fibers. The most typical practical use of complex preparations of acidic and weakly acidic proteases and xylanases is associated with the intensification of biotechnological processes in the fermentation industries in the production of alcohol, beer, with the processing of plant materials with a high content of protein substances and xylans in the production of feed and in other areas of the agricultural sector. In this regard, the selection of new fungal strains to obtain acidic proteases and xylanases is relevant.

Known strains are producers of fungal proteases from the genus Aspergillus oryzae 251-90, 824-32 (1, 2), during deep cultivation of which acidic and weakly acidic proteases are synthesized. The disadvantage of these producers is their low protease productivity and lack of biosynthetic ability in relation to xylanase.

Closest to the claimed object is a strain of Aspergillus oryzae 387, registration number VKPM F-683 (3), one of the most active producers of proteases. In the deep culture of the known strain Asp.oryzae 387 for 42 hours of growth, the proteolytic activity reaches 8.7-11.8 units. PS / cm ³ depending on the composition of the nutrient medium; in addition, xylanase with an activity of 3.4-4.3 units is synthesized. KS / cm ³ .

The disadvantage of this strain is the low level of proteolytic and xylanolytic activity of the culture fluid (short).

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 of proteases that exhibit maximum activity in the acidic and weakly acidic pH zones, as well as the synthesis of xylanase.

The technical result obtained from the use of the new strain is to obtain an enzymatic complex with high proteolytic and xylanolytic activity in the culture fluid, the level of which exceeds the analog by 2.5-2.8 times.

The problem is solved by creating a new strain by selection and mutagenesis from the known strain Aspergillus oryzae 387 (4), capable of rapid growth on simple composition nutrient media and the active synthesis of extracellular proteases and xylanase.

The inventive strain of Aspergillus oryzae 12 obtained by multistage selection using effective methods of mutagenesis, deposited in VKPM under No. F-932.

As a result of mutagenesis, after repeated selection and screening of variants, a strain of Aspergillus oryzae 12 was obtained - a producer of acidic and weakly acidic proteases and xylanases, as well as a concomitant enzyme, α-amylase.

Studied the levels of production of proteases and xylanase in the culture fluid obtained by growing the inventive strain and known strains 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, xylanase according to Shomody-Nelson, amylolytic according to GOST 20264.4-89. Comparative indicators of the levels of proteolytic activities of the culture fluid of strain Asp. oryzae 12 with prototypes are presented in tables 1 and 2.

Thus, as a result of selection and mutagenesis, a new strain of Aspergillus oryzae 12 was obtained, which has a high level of synthesis of proteases and xylanase, as well as α-amylase, which improves the manufacturability of the complex of enzymes of acidic and weakly acidic proteases and xylanase with a high level of their activity in culture liquids. The strain differs from the known and cultural-morphological characteristics.

The strain is stored in the form of freeze-dried culture and on mowing with wort 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 12 is characterized by the following properties.

Cultural signs. After 6 days of growth on the wort agar, colonies with a diameter of 60 × 65 mm are formed, the surface of the colony is rough with concentric circles, the profile of the colony is flat, the shape of the colony is irregular, the edges are wavy, the color is green, the edges are light yellow, the color of the colony darkens with age. The color on the back is gray.

When growing on Chapek’s agar medium, the colony size after 6 days is 25 × 25 mm, the colony color is olive, the shape of the colonies is round, the edges are even, light yellow.

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. Fungal mycelium septate, branched, with swelling, conidia are formed exogenously; the surface of conidia is smooth, the shape is mainly rounded; the thickness of the hyphae is 3-5 microns.

Physiological and biochemical characteristics. The type of catabolism is breathing. Attitude to oxygen - aerob. The optimum growth temperature is 30-34 ° 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. The producer grows on media with a solids content of from 0.5 to 18%.

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 12 was used to obtain acidic and weakly acidic proteases and xylanases, as well as a concomitant enzyme, α-amylase.

In deep cultivation of the strain Aspergillus oryzae 12, depending on the composition of the nutrient medium, the proteolytic activity is 21.0-24.6 units. PS / cm ³ , xylanolytic - 7.5-9.8 units. KS / cm ³ , amylolytic - 13.5-14.4 units. AC / cm ³ .

The invention is characterized by the following examples.

Example 1. The strain Aspergillus oryzae 12 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 (medium 1), 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 40 hours. The activity of extracellular proteases is 21.2 units. PS / cm ³ , xylanases - 9.5 units. KS / cm ³ , α-amylase - 13.5 units. AC / cm ³ .

Example No. 2. The strain Aspergillus oryzae 12 is cultivated on a nutrient medium of the following composition,%: barley flour - 3.0; wheat bran - 3.0; D-xylose - 0.5; KH ₂ PO ₄ - 1.5; the rest is tap water (medium 2), pH is natural. Cultivation is carried out as described in example 1. The activity of extracellular proteases is 23.0 units. PS / cm ³ , xylanases - 10.8 units. KS / cm ³ , α-amylase - 13.0 units. AC / cm ³ .

Example No. 3. The strain Aspergillus oryzae 12 is cultivated on a nutrient medium of the following composition,%: barley flour - 3.0; wheat bran - 3.0; xylan - 0.1; KH ₂ PO ₄ - 1.5; the rest is tap water (medium 3), the pH is natural. Cultivation is carried out as described in example 1. The activity of extracellular proteases is 24.6 units. PS / cm ³ , xylanases - 11.8 units. KS / cm ³ , α-amylase - 14.4 units. AC / cm ³ .

Table 1
Comparative evaluation of the physiological activity of Aspergillus oryzae fungi - producers of proteases Strain Duration of cultivation, h Enzymatic activity, units / cm ³ PS The cop AC 251-90 72 5,0 0 8.7 387 42 11.8 4.0 10.8 12 38 24.2 10,2 13.6 table 2
The influence of culture media on the enzymatic activity of the fungus Aspergillus oryzae pc. Strain Nutrient medium Enzymatic activity PS The cop AC units / cm ³ % increase units / cm ³ % increase units / cm ³ % increase 387 one 8.7 - 3.4 - 12.5 - 2 9.8 - 3.8 - 12.3 - 3 9.6 - 4.4 - 13,2 - 12 one 21,2 143 9.5 179 13.5 8 2 23.0 145 10.8 184 13.0 6 3 24.6 156 11.8 168 14,4 9

Sources of literature

1. 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.

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

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

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

Claims (1)

The strain of the fungus Aspergillus oryzae 12, VKPM F-932 - producer of acidic proteases and xylanases.