RU2303057C1 - Strain of mycelial fungus aspergillus aculeatus as producer of carbohydrases complex containing xylanases, beta-glucanases, pectinases and xyloglucanases - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymes.

SUBSTANCE: invention proposes the strain Aspergillus aculeatus VKM F-3766D that shows ability to produce complex of carbohydrases involving cellulases, beta-glucanases, xylanases, mannanases and pectinases and comprising enzymes with the high activity level. Invention provides possibility for preparing the complete complex of enzymes used in hydrolysis of vegetable raw polysaccharides and, if necessary, separate individual enzymes (components) of the complex. The complex and expensive nutrient media are not required for providing the high productivity of the strain. Invention can be used in different branches of alcoholic and food industry.

EFFECT: valuable properties of fungus.

5 ex

Description

The invention relates to the field of biotechnology and can be used in the microbiological industry, in the pulp and paper industry, in the food and alcohol industries, in brewing, as feed additives, for silage of feed in agriculture.

The closest in technical essence to the present invention are strains of the fungus Aspergillus niger, when deep cultivated on media with cellulolytic enzyme inducers, a complex of enzymes is obtained, including cellulase, xylanase, beta-glucosidase, beta-xylosidase, beta-glucanase and laminarinase (RF Patent No. 2057179, CL C12N9 / 42, 1996). The activity of enzymes in the culture fluid are, respectively: cellulase - 5.0 U / ml; xylanase - 125.0 U / ml; β-glucosidase - 20.0 U / ml; β-xylosidase - 36.0; β-glucanase - 0.95 U / ml and laminarinase - 1.25 U / ml.

However, despite the fact that fungi of the genus Aspergillus form enzyme complexes with a wide spectrum of carbohydrases, the activity of most of the individual components of the complex in Asp.niger strains indicated in the invention is not high enough, which makes their practical use impractical.

The technical problem to which the present invention is directed is to obtain a new highly active strain of mycelial fungi - a producer of a multienzyme complex containing a spectrum of carbohydrases, cleaving non-starch polysaccharides, and free from cellulases or containing low cellulase activity.

The technical result that can be obtained by carrying out the invention is to increase the processing speed of cellulose and hemicellulose-containing substrates when using the proposed strain.

To achieve the technical result, it is proposed to use a strain of the mycelial fungus Aspergillus aculeatus Iizuka Aj 79-178, a producer of the carbohydrase complex containing cellulases, beta-glucanase, xylanase, pectinase and mannanase.

The strain of the fungus Asp.aculeatus Iizuka Aj 79-178 was deposited in the All-Russian collection of microorganisms at the Institute of Biochemistry and Physiology of Microorganisms named after G.K.Skryabin RAS under the number VKM F-3766D.

The strain is obtained using multistage classical mutagenesis and selection from the original culture of Asp.aculeatus BKM F-3743D. A suspension of spores of the initial strain obtained in distilled water with the addition of 0.1% Tween-80 is irradiated with ultraviolet light (irradiation with a light flux with a power of 3 m W / cm ² ) for 3 min. Irradiated spores are plated on Petri dishes with selective media based on Hetchinson medium supplemented with 0.5% carboxymethyl cellulose (CMC), xylan, pectin, or xyloglucan, cultured at 28 ° C for 2 days, and Congo Red is stained. Mutants with improved production are selected visually for the increased areas of enlightenment around the colonies. The most active mutants selected on the plates are tested for the productivity of the synthesis of cellulases, β-glucanases, xylanases, pectinases and xyloglucanases when cultured in a liquid medium in flasks. The most active variants for β-glucanase, xylanase, pectinase and xyloglucanase activities and the least active for cellulase (CMC-ase) activity, selected during flask cultivation, are again (repeatedly) irradiated and selected on dishes and flasks, as described above.

Storage conditions: the strain can be stored in a lyophilized state for several years and / or on shoals with agarized Chapek’s medium or wort-agar at + 4 ° С with obligatory reseeding at least once for 3-6 months.

Cultural and morphological characteristics of the strain.

Colonies on Capek-agar with yeast extract (CYA) at a cultivation temperature of 25 ° C have a diameter of 35-37 mm on day 7; slightly radially borozdachatye, the surface is slightly velvety to leathery, the edge is uneven, the color of the colonies is white, the exudate is absent, the back side is ocher-yellow, the color in the conidial region is brown; the texture is dense, even, powdery; cut open radially folded, dull yellow; aerial mycelium is dense, white.

Colonies on Capek-agar with yeast extract and 20% sucrose (CY20S) at a cultivation temperature of 25 ° C have a diameter of more than 27-33 mm on the 7th day of growth; smooth, the edge is uneven up to 5 mm, the color is light brown in the conidial region; the texture is dense, even, woolly until powdery in the center of the colonies; reverse is even, pale yellow; dense aerial mycelium, white; exudate is absent, the reverse side is unpainted.

Colonies on Maltz-extract-agar (MEA) at a cultivation temperature of 25 ° C have a diameter of 42-45 mm on the 7th day of growth; slightly radially furrowed, the surface is woolly, the edge is uneven, the colony is black-brown; the texture is dense, even, powdery; the reverse is slightly radical folded, dull yellowish-brown; exudate is absent, aerial mycelium is sparse, white, the reverse side is unpainted.

Colonies on Capek-agar with yeast extract (CYA) at a cultivation temperature of 37 ° C have a diameter of 60 mm on the 7th day of growth.

Microscopic characteristics.

Reduced columnar conidial heads; are formed on CYA and MEA; on the CY20S medium, sporulation is absent. Conidiophores are slightly colored, in the area of apical expansion to brownish, smooth-walled 100-500 (2000) × 6.0-12.0 μm, apical extensions spherical, thick-walled ((28.8) -35-65 (97.2) (average value 52.1) μm in diameter). Falids are single-tier 4.5–9.0 × 2.7–4.8 μm, formed along the entire surface of the apical extension. On conidiophores only spherical apical extensions of 15-35 μm in diameter with sterigm primordia less than 1 μm are formed, conidia are absent.

When cultivated in deep conditions using soluble substrates (glucose, fructose, lactose), a loose branched mycelium with weak pelletization is formed, the specific initial mycelial growth rate was 0.25 h ^-1 , at the end of cultivation, 0.1-0.15 h ^-1 .

Physiological and biochemical characteristics of the strain.

Mesophilen. The optimum mycelium growth temperature is 32 ° C (29-34 ° C), the optimum for cellulase formation is 28 ° C (26-29 ° C). The optimal pH of growth and secretion of cellulases is 3.5-5.0. The growth of mycelium is also observed at pH 2.5, but a very weak formation of carbohydrase class enzymes is observed.

Nystatin resistance is weak. With surface cultivation, it is resistant to concentrations up to 0.5 μg / ml, at a concentration of 2.5 μg / ml, growth is completely inhibited. When digitonin (3.5-4.0 μg / ml) or Bengal pink (30-50 μg / ml) is added to the medium, the colony size is greatly reduced.

It is a prototroph. It is able to assimilate glucose, lactose, glycerin, galactose, xylose, D-mannitol, mannose, trehalose, L- and D-arabinose, sorbose, sorbitol, ribose.

Does not assimilate: L-ramnose, D-glucosamine, deoxyribose, deoxygalactose, 2-deoxy-D-glucose, 5-thio-D-glucose.

Uses ammonium and organic nitrogen, assimilates the nitrate form of nitrogen worse.

It forms enzyme systems that allow growing on the corresponding complex substrates: starch, xylan, laminarin, beta-glucan, lichenin, galactomannan, pectin, xyloglucan.

The catabolite repression of the biosynthesis of carbohydrases is significantly reduced. Verification of the catabolite repression of the biosynthesis of carbohydrase is as follows. Conidia are subcultured in test tubes with a minimal medium containing mineral salts, a trace amount (0.5 g / l) of yeast extract, xylan or pectin, as well as the test repressor or antimetabolite (glucose, 2-deoxy-D-glucose, lactose, glycerin and other). The diameter of the test tube is 9 mm, the height of the agar column is 50-60 mm. The tube is incubated for 4 days at 30 ° C and then 20 hours at 45 ° C. The stability of the carbohydrase biosynthesis to catabolite repression is judged by the depth of the xylan or pectin destruction zone (by the size of the clarification zone of the agar column in vitro) in the presence of a repressor or antimetabolite).

The obtained mutant Asp.aculeatus Aj 79-178 (F-3766D) in its morphological characteristics when growing on glucose-potato agar, on sporulation agar (CM agar) differs from the original strain Asp.aculeatus F-3743D) in a reduced sporulation rate, the color of the spore pigment and colony coloration (white instead of black and black) on the reverse side when grown on agarized media, slower growth on solid media, enhanced ability to deep biochemically cultivate various carbohydrases - β-glucanases, xylanases, pectin and xyloglucanases, while not forming or forming a very small amount of cellulase.

This type of mycelial fungus is not listed as pathogenic in the "Regulation on the procedure for recording, storing, handling, dispensing and sending cultures of bacteria, viruses, rickettsia, fungi, protozoa, mycoplasmas, bacterial toxins, poisons of biological origin."

The cultivation of the strain Asp.aculeatus Aj 79-178 (F-3766D) is carried out under aerobic conditions in a submerged state on a nutrient medium containing one or more substrates - carbon sources, which are the inducers of enzyme biosynthesis. Non-inductor substrates can also be used as substrates. The strain is capable of secreting a complex of enzymes - carbohydrases (β-glucanases, xylanases, pectinases and xyloglucanases, etc.) in the culture medium under appropriate conditions of the cultivation process based on the use of soluble substrates, for example glucose, and in the presence of inducer sources (xylose, various plant waste) ) Glucose in the cultivation medium can be replaced by a cheaper product - starch hydrolyzate.

The activity of β-glucanases, xylanases, xyloglucanases, pectinases and cellulases (CMC-basics) in the culture fluid is determined by the ability to cleave. β-glucan, xylan, xyloglucan, polygalacturonic acid and CMC, respectively. For a unit of β-glucanase, xylanase, xyloglucanase, pectinase and CMC-ase activity, one takes such a number of enzymes that within 1 min at a temperature of 50 ° C and pH 5.0 it releases 1 μmol of reducing sugars equivalent to 1 μmol of glucose and determined by the Somoji method -Nelson (A.P. Sinitsyn, A.V. Gusakov, V.M. Chernoglazov. Bioconversion of lignocellulosic materials. Textbook, Moscow: Publishing House of Moscow State University, 1995, p.144-156).

Enzyme preparations obtained using the proposed strain can be used in the form of a culture fluid, in the form of concentrated concentrated preparations obtained by ultrafiltration or evaporation of a culture fluid, or in the form of dry preparations obtained by drying or granulation.

The possibility of using the invention is illustrated by examples, which do not limit the scope and essence of the claims associated with them.

Example 1. Fermentation in rocking flasks. To obtain seed (inoculum), the culture of the fungus Asp.aculeatus Aj 79-178 (F-3766D) is grown on wort or glucose-potato agar at 29 ° C for 4 days and then at room temperature in the light for 5 days . Inoculation of flasks is carried out with 1 ml of a suspension of spores washed with agar with water containing 0.1% Tween-80. The cultivation of the strain is carried out under aerobic conditions in Erlenmeyer rocking flasks with a volume of 750 ml, containing 100 ml of a liquid medium of the following composition, in g / l: beet pulp - 10.0; malt sprouts - 30.0; (NH ₄ ) ₂ SO ₄ - 3.0; NaNO ₃ - 3.0; KH ₂ RO ₄ - 4.0; MgSO ₄ × 7H ₂ O - 0.5; pH 4.5-5.0. The flasks are incubated on a shaker at 29 ° C and 200 rpm for 132-144 hours

The activities of xylanases, pecginases, β-glucanases, xyloglucanases and cellulases are 450, 50, 150, 7 and 0.8 units / ml, respectively, which is not worse than the initial strain by cellulase activity and higher by 200%, 350%, 150%, 200 % and 150% in the activity of xylanases, pectinases, β-glucanases and xyloglucanases.

Example 2. Fermentation of a strain of Asp.aculeatus Aj 79-178 (F-3766D) in rocking flasks. The cultivation is carried out in Erlenmeyer rocking flasks, as described in example 1, using a liquid nutrient medium of the following composition, in g / l: beet pulp - 10.0; malt sprouts - 15.0; (NH ₄ ) ₂ SO ₄ - 3.0; NaNO ₃ - 3.0; KH ₂ PO ₄ - 4.0; MgSO ₄ × 7H ₂ O - 0.5; pH 4.5-5.0. The flasks are incubated on a shaker at 29 ° C and 200 rpm for 132-144 hours

The activities of xylanases, pectinases, β-glucanases, xyloglucanases and cellulases are 270, 95, 150, 10 and 0.6 units / ml, respectively, which is not worse than the initial strain by the activity of cellulases and higher by 150%, 300%, 130% and 250 % activity of xylanases, pectinases, β-glucanases, xyloglucanases.

Example 3. The cultivation of the strain Asp.aculeatus Aj 79-178 (F-3766D) is carried out in Erlenmeyer rocking flasks, as described in example 1, using a liquid nutrient medium of the following composition, in g / l: ground oats - 15; malt sprouts - 15; (NH ₄ ) ₂ SO ₄ - 3.0; NaNO ₃ - 3.0; KH ₂ RO ₄ - 4.0; MgSO ₄ × 7H ₂ O - 0.5; pH 4.5-5.0. The flasks are incubated on a rocking chair at 29 ° C and 200 rpm for 144 hours.

The activities of xylanases, pectinases, β-glucanases, xyloglucanases and cellulases are 670, 24, 160, 15 and 0.3 units / ml, respectively, which is not worse than the initial strain by cellulase activity and higher by 450%, 140%, 250% and 300 % activity of xylanases, pectinases, β-glucanases and xyloglucanases.

Example 4. Fermentation of a strain of Asp.aculeatus Aj79-178 (F-3766D) in a fermenter. The cultivation process is carried out in an ANKUM 2M type fermenter with a total volume of 10 l and a working volume of 6 l. Aeration is 1 volume of air per 1 volume of medium in the fermenter. The fermenter is inoculated with 500 ml of vegetative mycelium obtained after 48 hours of cultivation at 30 ° C on Erlenmeyer rocking flasks containing the medium of the following composition, in g / l: glucose - 10.0; yeast extract - 5.0; (NH ₄ ) ₂ SO ₄ - 3.0; NaNO ₃ - 3.0; KH ₂ PO ₄ - 4.0; MgSO ₄ × 7H ₂ O - 0.5; pH 4.5-5.0.

The first phase of cultivation (in which the fungus mainly grows and accumulates biomass) is carried out for 48 hours at 30 ° C and pH 4.0. The composition of the nutrient medium: beet pulp - 10.0; malt sprouts - 20.0; (NH ₄ ) ₂ SO ₄ - 3.0; NaNO ₃ - 3.0; KH ₂ RO ₄ - 4.0; MgSO ₄ × 7H ₂ O - 0.5.

After the consumption of soluble sugars, which is usually observed after 48 hours and which is judged by an increase in pO ₂ , the second phase of cultivation begins (during which enzymes accumulate in the growth medium). In the second phase of fermentation (extracellular enzyme biosynthesis phase), an aqueous solution of carbohydrates with a total concentration of 50%, consisting of a mixture of glucose: xylose (4: 1) is continuously added to the fermenter so that the total concentration of carbohydrates in the nutrient medium in the fermenter does not exceed 1 g / l The initial feed rate of the mixture is 3 ml / l / h.

The activity of xylanases, pectinases, β-glucanases, xyloglucanases and cellulases for 144 hours of cultivation is 1700, 110, 250, 50 and 0.8 units / ml, respectively, which is not worse than the initial strain by cellulase activity and higher by 750%, 450%, 350% and 500% in the activity of xylanases, pectinases, β-glucanases and xyloglucanases.

Example 5. Fermentation of a strain of Asp.aculeatus Aj 79-178 (F-3766D) in a fermenter. The cultivation process is carried out in an ANKUM 2M type fermenter with a total volume of 10 l and a working volume of 6 l. Aeration is 1 volume of air per 1 volume of medium in the fermenter. Inoculate 500 ml of vegetative mycelium obtained after 36 hours of cultivation on Erlenmeyer rocking flasks (as in Example 4).

Use the nutrient medium of the following composition, in g / l: ground oats - 20; malt sprouts - 20; glucose - 16.0; xylose - 4.0; (NH ₄ ) ₂ SO ₄ - 3.0; NaNO ₃ - 3.0; KH ₂ PO ₄ - 4.0; MgSO ₄ × 7H ₂ O - 0.5.

The first phase of cultivation (in which the fungus mainly grows and accumulates biomass) is carried out for 46 hours at 30 ° C and pH 4.0.

In the second phase of fermentation (extracellular enzyme biosynthesis phase), an aqueous solution of carbohydrates with a total concentration of 25%, consisting of a mixture of glucose: xylose (10: 1) is continuously added to the fermenter so that the total concentration of carbohydrates in the nutrient medium in the fermenter does not exceed 1 g / l The initial feed rate of the mixture is 3 ml / l / h.

The activity of xylanases, pectinases, β-glucanases, xyloglucanases and cellulases for 144 hours of cultivation is 1300, 25, 450, 45 and 0.5 units / ml, respectively, which is not worse than the initial strain by cellulase activity and higher by 400%, 200%, 500% and 450% of the activity of xylanases, pectinases, β-glucanases and xyloglucanases.

Thus, the proposed strain Aspergillus aculeatus Aj 79-178 (F-3766D) has the ability to produce a complex containing enzymes of a high level of activity and consisting of carbohydrase-xylanases, pectinases, β-glucanases and xyloglucanases and practically devoid of cellulases. This makes it possible to obtain a complete complex of enzymes for the hydrolysis of non-starch polysaccharides of plant materials, as well as, if necessary, individual individual enzymes (components) of the complex.

To achieve high productivity of the strain does not require the use of complex and expensive nutrient media. For cultivation, nutrient media traditionally used in industrial technologies for producing such enzyme preparations can be used.

The preparations obtained on the basis of the proposed strain can significantly increase the efficiency and expand the range of use of enzyme preparations in various fields of biotechnology and, especially, as feed additives.

Claims (1)

The strain of mycelial fungus Aspergillus aculeatus BKM F-3766D is a producer of a complex of carbohydrases containing xylanases, pectinases, xyloglucanases and β-glucanases and exhibiting low cellulase activity.