Fungal strain aspergillus foetidus - producer of complex of pectinases, β-glucanase, xylanase, cellulase, chitinase, mannanase and protease for destruction of polymers of plant and microbial raw material

FIELD: biotechnology.

SUBSTANCE: strain of micromycete Aspergillus foetidus 379-K-5-1 is proposed, producing a complex of pectinases, β-glucanase, xylanase, cellulase, chitinase, mannanase and protease for the destruction of polysaccharides of a plant and microbial material. The strain is deposited in the Departmental collection of useful microorganisms for agricultural purposes of the Russian Academy of Agricultural Science (RCAM) of the State Scientific Institution of the Russian National Research Institute for Agricultural Microbiology under the registration number of RCAM 01136. The advantage of the new strain is to obtain the more active complex of enzymes catalyzing the hydrolysis of polysaccharides, as well as endopolygalacturonase and pectinesterase. The strain Aspergillus foetidus RCAM 01136 is produced using efficient methods of selection and mutagenesis using UV irradiation and nitrosoguanidine from the known strain.

EFFECT: invention enables to expand the range of the enzymes obtained, used for the destruction of polysaccharides.

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The invention relates to biotechnology. The strain of the fungus Aspergillus foetidus 379-K-5-1 produces a set of hydrolytic enzymes and deposited in the collection of useful microorganisms for agricultural purposes wildebeest ARRIAM number Cam system for true 01136. The invention provides an active synthesis of complex enzymes: pectinase, β-glucanase, the intended cellulases, chitinases, mannanase and protease used in the food industry for the destruction of plant substrates and microbial biomass, with the aim of obtaining biologically active additives with prescribed functional properties.

Strain Aspergillus foetidus 379-K-5-1 (Cam system for true 01136) obtained using effective methods of selection and mutagenesis with the use of UV irradiation and nitrosoguanidine (concentration of 0.3%, exposure time 20 minutes) from a known strain of Aspergillus foetidus 379-K (VKPM F-962) used for deep cultivation in the production of pectinase.

The invention provides a complex enzyme preparation with broad substrate specificity for the deep destruction of plant and microbial raw materials based on the depth of the cultivation of new strain - producer of hydrolytic enzymes.

The enzymes that catalyze the hydrolysis of polysaccharides, are of great industrial importance in the processing industries the food industry to implement bioca�litijska processes ensure the efficient degradation of plant polymers and microbial raw materials in the technology of production of juices, fruit drinks, extracts and biologics food and feed purposes. In this regard, breeding new strains - producers of a complex of highly active hydrolases: pectinase, β-glucanase, the intended cellulases, chitinases, mannanase and proteases, are highly relevant.

Known producing strains of the complex hydrolases containing the intended β-glucanase, pectinases and xyloglucans of the genus Aspergillus is Aspergillus aculeatus [1]. The disadvantage of this producer is the absence of secreted them chitinolytic, proteolytic and mennonitechurch enzymes.

Known strain-producer of cellulases, β-glucanase and xylanases from the sort - of Trichoderma is Trichoderma longibrachiatum [2]. The disadvantage of this producer is the absence of secreted them pectolytic, chitinolytic and proteolytic enzymes.

Closest to the claimed object is a strain of Aspergillus foetidus 379-It is one of the most active producers of pectinase, registration number VKPM F-962 (Patent RF№2393212, 2010) [3].

The disadvantage of this strain is the low level of activity of β-glucanase, chitinases and mannanase required for microbial degradation of raw materials.

The problem posed by the present invention is to obtain strains of micromycetes Aspergillus foetidus,�latausha high ability to form complex hydrolytic enzymes: pectinase, β-glucanase, the intended cellulases, chitinases, mannanase and protease.

The technical result obtained from the creation and use of a new strain of Aspergillus foetidus 379-K-5-1 (Cam system for true 01136), is to obtain a more active complex of enzymes that catalyze the hydrolysis of polysaccharides, the level of activity which in the 2.0-4.0 times higher than analog and also andprecautions and pectinesterase, the level of activity in the culture fluid exceeds the performance of analog 1.5 times, and protease, exceeding the performance of analog 1.2 times.

The essence of the present invention - to provide a new strain - producer of the highly complex hydrolases: pectinase, β-glucanase, the intended cellulases, chitinases, mannanase and protease.

The problem is solved by the creation of new producer strain complex hydrolases by breeding and mutagenesis combined (chemical and physical) from a known strain of Aspergillus foetidus 379-K, is able to synthesis complex of hydrolytic enzymes necessary for degradation of microbial and plant materials.

The inventive strain of Aspergillus foetidus 379-K-5-1 (Cam system for true 01136) obtained by multistep selection with the use of effective methods of mutagenesis using UV irradiation and nitrosoguanidine.

Activity hemicellulolytic enzymes was determined by the method of Somodi-Also�and, based on measuring the rate of formation of reducing Sugars during the hydrolysis of the substrate, as a result of the action of enzymes (3). Activity andprecautions and pectinesterase GOST 20264.3-81. Comparative indicators of levels of enzyme activity in the culture fluid of strain Aspergillus foetidus 379-K-5-1 with the prototype presented in table 1.

Thus, through selection and combination of mutagenesis obtained new active strain of Aspergillus foetidus 379-K-5-1 (Cam system for true 01136) with a high level of synthesis of complex hydrolases, including pectinase, β-glucanase, the intended cellulases, chitinases, mannanase and proteases, which improves process efficiency in the hydrolysis of plant and microbial raw materials for the purpose of obtaining complex food additives.

Table 1
Comparative indicators of the level of enzyme activity in the culture fluid of fungi Aspergillus foetidus 379-K and Aspergillus foetidus 379-K-5-1
Enzyme activityAspergillus foetidus
PCs.379-KPCs.379-K-5-1
β-Glucanase, u β-GCS/cm3 6,5the 13.3
Cellulose, u of CA/s32,66,1
Silananda, ed KS/cm33,313,5
Mandanna, ed MS/cm30,270,64
Chitinase, u XC/cm30,0240,05
Proteasa, units PS/cm32,22,7
Endopolyploidy, ed Endo-PGS/cm32,013,7
Pectinesterase, ed PES/cm32,13,3

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

Strain Aspergillus foetidus 379-K-5-1 deposited in all-Russian Collection of Beneficial Microorganisms of agricultural�hosts-agricultural purposes (Cam system for true) wildebeest ARRIAM under a collector's room Cam system for true 01136.

Cultural morphological characteristics of the strain Aspergillus foetidus 379-K-5-1:

Culture is strongly branching mycelium separated by partitions. Conidia formed by micromycetes, smooth. The shape of the conidia round, rarely oval. Color black conidia, with abundant formation of aerial mycelium. Colonies with convex elevation in the center. Reverse side of colony brown. The surface of the colony folded.

On the environment wort-agar, colonies are round, to 5 days reach 37-38 mm, have Mozgovoy folding. The Central area of the colony - area abundant sporulation - diameter 12-14 mm black color with a dark brown shade and abundant formation of aerial mycelium. The edge of the colony is smooth. Reverse side of colony brown.

On the medium with maltose colonies are round, on the edge of the colony abundant mycelium, colony size on day 5 from 32 to 34 mm, the edge of the colony is smooth and even, colony structure coarse-grained, large colony. The surface of the folded colonies. The reverse side of the colony is dark gray.

On the medium with extract of malt sprouts and wheat bran colonies are round, throughout the colony of aerial mycelium, the edge of the colony is flat and smooth, the structure of fine-grained colony, colonies large colonies on day 5 - 38-40 mm. the Surface of the colony is strongly folded.

On environments� with starch colony of black color, form colonies round the edge of the smooth colonies, colony structure coarse-grained, large colony size, colony 5 days - 30 to 32 mm. the Reverse side of the colony is light brown.

Physiological and biochemical characteristics of the strain Aspergillus foetidus 379-K-5-1:

Strain assimilates nitrate and ammonium salt. Culture weakly absorbs the glucose. Well assimilated maltose, starch and sucrose.

The aerobic respiration. The optimum growth temperature of 28-30°C; the optimum pH value for growth and biosynthesis of the fungus from 5.3 to 5.5. The growth of a producer is in the area of pH from 2.7 to 3.0. Pathogenic properties do not possess.

Example 1. Strains of Aspergillus foetidus 379-K-5-1 and 379-cultured on a nutrient medium of the following composition: malt sprouts - 5,0%, wheat bran - 10,0%, (NH4)2SO4at 1.1%, KH2PO4- 0,5%, MgSO4- 0,5%; the rest is tap water, pH natural. The fermentation culture medium is seeded with spore sowing material in an amount of 2%, grown at 30°C for 120 hours. The mushroom cultivation is carried out in Erlenmeyer flasks with a volume of 750 cm3containing 100 cm3on a pie rocking chair 220 rpm at 30°C for 72 hours. The level of activity of the enzymes synthesized by the strains 379-K-5-1 and 379-K, were as follows: for β-glucanase is 12.4 IU/cm3and 5.7 u/cm3on xylanase is 8.7 u/cm3and 5.9 u/cm3, the cellulase - 6,0 u/cm3and 4.8 u/cm3on chitinase - 0,039 u/cm3and 0,018 u/cm3on mannanase - 0,57 u/cm3and 0,19 u/cm3on the protease - 2,4 u/cm3and 1.7 u/cm3on antipolysaccharide to 3.2 u/cm3and of 1.85 u/cm3on pectinesterase and 2.9 u/cm3and 1.73 u/cm3respectively.

Example 2. Strains of Aspergillus foetidus 379-K-5-1 and 379-cultured on a nutrient medium of the following composition: malt sprouts - 5,0%, wheat bran - 10,0%; (NH4)2SO4to 1.1%; KH2PO4- 0,5%; MgSO4- 0,5%; the rest is tap water, pH natural. Fermentation environment is seeded with spore sowing material in an amount of 2%, grown at 30°C for 120 hours. The mushroom cultivation is carried out in Erlenmeyer flasks with a volume of 750 cm3containing 100 cm3nutrient medium on rocking 220 rpm, at 30°C for 48 hours.

The level of activity of the enzymes synthesized by the strains 379-K-5-1 and 379-K, is: β-GCS - 9,0 u/cm3and 5.2 IU/cm3; COP of 13.5 u/cm3and 7.6 u/cm3; CMC - 6,1 u/cm3and 5.0 u/cm3, CH - 0,035 IU/cm3and 0.012 u/cm3on MS - 0,34 u/cm3and 0.10 u/cm3, PS-1.5 IU/cm3and 1.2 u/cm3by Endo-PGS - 3,7 u/cm3and 2,01 u/cm3by PES - 1,8 u/cm3and 1.02 u/cm3respectively.

Approx�R 3. Strain Aspergillus foetidus 379-K-5-1 and 379-cultured on a nutrient medium of the following composition: malt sprouts - 5,0%, wheat bran - 10,0%; (NH4)2SO4to 1.1%; KH2PO4- 0,5%; MgSO4- 0,5%; the rest is tap water, pH natural. Fermentation environment is seeded with spore sowing material in an amount of 2%, grown at 30°C for 120 hours. The mushroom cultivation is carried out in Erlenmeyer flasks with a volume of 750 cm3containing 100 cm3nutrient medium on rocking 220 rpm, at 30°C for 96 hours.

Maximum accumulation of β-glucanase was observed in strains of A. foetidus 379-K-5-1 to 96 hours of cultivation. The level of activity of the enzymes synthesized by the strains 379-K-5-1 and 379-K, were as follows: for β-glucanase and 13.3 u/cm3and 6.5 u/cm3on xylanase - 3,6 u/cm3and 1.56 u/cm3on the cellulase - 2,4 u/cm3and 0.89 u/cm3on chitinase - 0,05 u/cm3and of 0.024 u/cm3on mannanase and 0.64 u/cm3and 0.27 u/cm3on the protease - 2,7 u/cm3and 2.2 u/cm3on andprecautions of 2.6 u/cm3and 1.3 u/cm3on pectinesterase and 3.3 IU/cm3and 2.1 u/cm3respectively (table.2).

Table 2
The influence of the duration of the ku�of tipirovaniya on the physiological activity of micromycetes Aspergillus foetidus producers hydrolases
The strain of the fungusThe duration of cultivation, hEnzyme activity, units/cm3
β-GKSXCMSEndo-PGSPES
379-K485,20,0120,12,011,02
725,70,0180,191,851,73
966,50,0240,271,32,1
379-K-5-1489,00,0350,343,71,8
7212,40,0390,45 3,22,5
96the 13.30,050,642,63,3

Sources of information

1. Microbial enzymes and biotechnology. Under the editorship of V. M. Fogarty. - Moscow: Agropromizdat, 1986.

2. Gracheva, I. M., Krivov, A. Yu., Technology of enzyme preparations. - Moscow: Publishing House "Elevar", 2000.

3. GOST R 53973-2010 enzyme Preparations. Methods of determining β-glucanase activity.

4. Sinitsyn, A. P., Chernoglazov V. M., A. V. Gusakov Bioconversion of lignocellulosic materials. Training manual. Moscow: MSU Publishing house, 1995, pp. 144-156.

Similar patents

1. RF patent №2303057 C1, C12N 1/14, C12N 9/42. The strain of the filamentous fungus Aspergillus aculeatus - producer of complex carbohydrat containing the intended beta-glucanase, pectinases and xyloglucans.

2. RF patent №2303065 C1, C12N 9/42, C12N 1/14. The strain of the filamentous fungus Trichoderma longibrachiatum producer of cellulases, beta-glucanase and xylanases.

3. RF patent №2393212 C1, C12N 1/20, C12N 9/24. The strain of the fungus Aspergillus foetidus 379-To - producer pectolytic enzymes.

The strain of the fungus Aspergillus foetidus 379-K-5-1, deposited in the Departmental collection of useful microorganisms for agricultural purposes RAAS under registration number Cam system for true 01136 - producer.�sa pectins, β-glucanase, the intended cellulases, chitinases, mannanase and protease for the degradation of polymers of plant and microbial material.



 

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