Fungus strain aspergillus oryzae as producer of acid proteases and xylanase

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

 

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

The invention relates to biotechnology, in particular to the creation of a new strain used to obtain the complex of enzymes, acid and weakly acidic protease, xylanase. Proteolytic and xylanolyticum enzymes are widely used in various industries: alcohol, beer, wine, food and others. This is because processed agricultural raw materials contains high molecular weight protein polymers and vegetable fibers. The most typical practical application of complex products, acidic and weakly acidic proteases and xylanases due to the intensification of biotechnological processes in the fermentation industries for production of alcohol, beer, processing of vegetable raw materials with a high content of proteins and Xylenol in feed production and in other areas of agriculture. In this regard, the selection of new fungal strains for the floor is to be placed acidic proteases and xylanases relevant.

Known strains - producers of fungal proteases of the genus Aspergillus oryzae 251-90, 824-32 (1, 2), with deep cultivation which are synthesized acidic and weakly acidic protease. The disadvantage of these producers is their low productivity by proteases and the lack of biosynthetic capacity in relation to the 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. Deep culture known strain Asp.oryzae 387 42 hours growth of proteolytic activity reaches of 8.7 and 11.8 units PS/cm3depending on the composition of the nutrient medium; in addition, synthesized xylanase activity of 3,4-4,3% CS/cm3.

The disadvantage of this strain is the low level of proteolytic and xylanolyticum activity in the culture fluid (k.j).

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 maximum activity in the acidic and weakly acidic zone pH, as well as to the synthesis of xylanase.

The technical result from the use of a new strain is getting enzymatic complex with high proteolytic and xylanolyticum activity in the culture fluid,the level of which exceeds the similar 2.5-2.8 times.

The problem is solved by the creation of new strains through breeding and mutagenesis of a known strain of Aspergillus oryzae 387 (4), capable of rapid growth on simple nutrient media and active synthesis of extracellular protease and xylanase.

The inventive strain of Aspergillus oryzae 12 obtained by multi-stage selection using effective methods of mutagenesis, deposited in ACIM # F-932.

As a result of mutagenesis after repeated selection and screening of alternatives was obtained a strain of Aspergillus oryzae 12 producing acidic and weakly acidic protease and xylanase, and concomitant enzyme - α-amylase.

Studied the levels of production of protease and xylanase 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 in optimal conditions, the action of proteases, xylanase on the Somogyi-Nelson, amylolytic - according to GOST 20264.4-89. Comparative indicators of levels of proteolytic activity of the culture liquid strain Asp. oryzae 12 prototypes presented in table 1 and 2.

Thus, through selection and mutagenesis received a new strain of Aspergillus oryzae 12, with a high level of sin is ESA protease and xylanase, and α-amylase, which improves the manufacturability of the process of obtaining enzyme complex acidic and weakly acidic protease and xylanase with high level of their activity in the culture fluid. Strain differs from known and cultural-morphological characters.

The strain is stored in a lyophilized culture and on) 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 12 is characterized by the following properties.

Cultural characteristics. After 6 days of growth on wort-agar formed colonies with a diameter of 60×65 mm, the surface is 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 25×25 mm, color olive colony, the colonies are round, smooth edge, light yellow.

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

Morphology of strain. The fungal mycelium septate, branched, with swellings, conidia are formed acsog the NGOs; the surface of conidia smooth, shape, mostly rounded; height hyphae 3-5 ám.

Physiological and biochemical characteristics. Type catabolism - breath. Relation to oxygen - aerobe. Optimum temperature for growth 30-34°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. The producer is growing on media with solid content of 0.5 to 18%.

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 12 used to get acidic and weakly acidic protease and xylanase, and concomitant enzyme - α-amylase.

In submerged cultivation of a strain of Aspergillus oryzae 12 depending on the composition of the nutrient medium proteolytic activity is 21,0-24,6 units PS/cm3, xylanolyticum is 7.5 and 9.8% CS/cm3, amylolytic to 13.5-14.4V unit ° C/cm3.

The invention is characterized by the following examples.

Example 1. A strain of Aspergillus oryzae 12 are 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 (environment 1), pH - nature is hydrated. 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 40 hours. The activity of extracellular proteases is 21.2 units PS/cm3, xylanase to 9.5% CC/ cm3that α-amylase to 13.5 unit ° C/cm3.

Example No. 2. A strain of Aspergillus oryzae 12 are cultivated in a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran - 3,0; D-xylose and 0.5; SC2PO4to 1.5; the rest is water (medium 2), pH natural. The cultivation is carried out as described in example 1. The activity of extracellular proteases is 23,0 units PS/cm3, xylanase and 10.8% CS/cm3that α-amylase - 13,0% AU/cm3.

Example No. 3. A strain of Aspergillus oryzae 12 are cultivated in a nutrient medium of the following composition, %: barley flour - 3,0; wheat bran - 3,0; xylan - 0,1; KN2PO4to 1.5; the rest is water (medium 3), pH natural. The cultivation is carried out as described in example 1. The activity of extracellular proteases is 24,6 units PS/cm3, xylanase and 11.8% CS/cm3that α-amylase - 14.4V unit ° C/cm3.

Table 1

Comparative evaluation of physiological activity from Aspergillus oryzae producing proteases
StrainDuration of cultivation, hEnzymatic activity, units/cm3
PSCAAC
251-90725,008,7
3874211,84,010,8
123824,210,213,6
Table 2

Effect of nutrient media on the enzymatic activity of the fungus Aspergillus oryzae St and St
StrainNo. nutrient mediumEnzymatic activity
PSCAAC
units/cm3% increase units/cm3% increaseunits/cm3% increase
38718,7-3,4-12,5-
29,8-the 3.8-12,3-
39,6-4,4-13,2-
121of 21.21439,517913,58
223,014510,818413,06
324,615611,816814.4V9

References

1. Zueva W. Physiological and biochemical IP is to study experimentally obtained mutant Aspergillus oryzae - active producer of acid proteases. Abstract of Cand. the dissertation. - M.: 1971.

2. AS the USSR №1440922, CL 12N 1/14, 1988.

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

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

The strain of the fungus Aspergillus oryzae 12, VKPM F-932 - producer of acid protease and xylanase.



 

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