RU2287570C2 - STRAIN OF MYCELIAL FUNGUS PENICILLIUM FUNICULOSUM AS PRODUCER OF CARBOHYDRASES COMPLEX CONSISTING OF CELLULOSES, β-GLUCANASES, XYLANASES, PECTINASES AND MANNANASES - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymology, agriculture, microbiology, food processing industry.

SUBSTANCE: invention relates to the strain of mycelial fungus Penicillium funiculosum VKM F-3668D prepared by multistep classic mutagenesis and selection methods from the parent culture of Penicillium funiculosum P1 (VKM F-3661D) and by using ultraviolet irradiation. The strain shows capacity for producing the complex of highly active carbohydrates involving celluloses, β-glucanases, xylanases, pectinases and mannanases that provides possibility for preparing the much more active and full enzyme complex and if necessary the separate individual enzymes (components) of this complex. For providing the high productivity of the strain nutrient media used conventionally by industrial technology for preparing similar enzyme preparations can be used instead using the complex and expensive nutrient media. Invention provides enhancing effectiveness and expanding assortment of enzyme preparations in different fields of biotechnology being as fodder supplements especially.

EFFECT: valuable properties of fungus strain.

4 ex

Description

The invention relates to the field of biotechnology and can be used in the microbiological and food industry and in agriculture. Carbohydrase multienzyme complexes containing β-glucanolytic, cellulose and hemicellulolytic enzymes that lyse the main components of the cell wall, as well as individual carbohydrases, can be used for biodegradation of cellulose and hemicellulose-containing substrates, including industrial and agricultural wastes, for plant cell wall biodegradation as well as in the food and alcohol industry, in the brewing industry, as additives in feed, for silage of feed in agriculture.

There are many known strains of producers of individual carbohydrases, mainly cellulases and methods for their cultivation under aerobic conditions.

Typically, microorganisms, in particular mycelial fungi, form a complex of enzymes that destroy plant substrates. As a rule, the complex includes mainly cellulases, as well as other carbohydrases, among which xylanases, β-glucanases and pectinases are most often found.

The choice of producer strain is determined by its ability to provide fairly high levels of carbohydrase activity in the fermentation medium, the rate of enzyme formation, the release of enzymes per unit mass of the substrate used, and the cost of the substrates themselves.

Aspergillus niger strains are known, when cultured on media with cellulolytic enzyme inducers, a complex of enzymes is obtained, including cellulase, xylanase, β-glucosidase, β-xylosidase, β-glucanase and laminarinase (RF Patent N 2057179, class C 12 N 9/42 , 1996). The activity of enzymes in the culture fluid are, respectively: cellulase - 5.0 U / ml; xylanases - 125.0 U / ml; β-glucosidase - 20.0 U / ml; β-xylosidase - 36.0 U / ml; β-glucanase - 0.95 U / ml and laminarinase - 1.25 U / ml. Although fungi of the genus Aspergillus form complexes with a wide spectrum of carbohydrases, the activity of most of the individual components of the complex is not high enough, which often makes their practical use impractical.

Active producers of cellulases are strains of mycelial fungi of the genus Trichoderma (US Patent N 4472504, class C 12 N 9/42, 1984, patent GDR N 291673, class C 12 N 9/42, 1991). Fungi of the genus Trichoderma, as a rule, have high productivity in relation to cellulases, but to a much lesser extent in relation to other carbohydrases (β-glucanases, xylanases, pectinases, mannanases), which are necessary for the hydrolysis of various polysaccharides that are part (in addition to cellulose) of plant raw materials.

Known mutant Trichoderma reesei BCM 18.2 / KK (VGNKI-28), obtained by parasexual processes from the original culture of Trichoderma reesei IMET 43803, which has increased productivity and allows for single-stage fermentation to provide high enzyme activity (cellulase) per unit mass of the substrate used (Patent Patent RF No. 2001949, C 12 N 9/42, 1993). Strain 18.2 / KK has enzyme systems that allow it to grow on a medium with cellulose, starch, chitin, pectin, xylan, laminarin, lichenin. When cultivated on a liquid nutrient medium based on beet pulp, an activity level of 3.5-4.2 u / ml FPA is achieved (cultivation time 110 h), when cultured in a fermenter on a medium with lactose 18.2 u / ml (81 h), when cultured in a fermenter on whey - 12-14 u / ml (100-110 h). (FPA is the activity of the cellulase complex, determined by filter paper and expressed in international units according to the IUPAC recommendation (T.K. Jose., Pure and Appl. Chem, vol. 59, No. 2, pp. 257-268).

Previously, we obtained, using multistage classical mutagenesis and selection from the original culture of Trichodrema reesei QM 6a, a mutant Trichoderma longibrachiatum TW-1 (BKM F-3634 D), (application for the grant of patent of the Russian Federation No. 2001105482/13 of 02.28.2001), which, when cultured in a liquid medium based on microcrystalline cellulose (MCC) and lactose (cultivation time 120 h), provides the activity of cellulases, β-glucanases, xylanases, pectinases and mannanases, respectively: 6.0; 30.2; 22.0; 7.5 and 1.2 u / ml of culture fluid; when cultured in a fermenter on an environment with wheat bran, starch hydrolyzate and MCC - 20.0; 380.0; 65.0; 23.0 and 24.0 units / ml, respectively (cultivation time 120 h).

The ability to produce a complex of carbohydrases is also possessed by fungi of the genus Penicillium. The literature describes a mutant Penicillium funiculosum Cu-1, which on cellulose, wheat or rice bran media forms cellulases - 2.6-3.3 units / ml FPA, β-glucosidase - 3.0-7.5 units / ml and xylanase - 33- 36 units / ml. (Enzyme Microb. Technol., 1986, vol. 8, p. P. 105-108).

There is evidence of the production of a complex of carbohydrases with a wide range of activities (cellulolytic, hemicellulolytic, glucanolytic, amylolytic, pectinolytic and proteolytic) by Penicillium janthinellum V39 / S fungi (DE Patent No. 4243423, July 1, 1993). There is no information on the magnitude of the activities.

Known mutant Penicillium occitanis Pol6, which has a fairly high productivity of a wide range of carbohydrases on media with pure cellulose or in combination with wheat bran, beet pulp or xylan (Enzyme Microb. Technol., 1990, vol.12, pp691-696). in liquid nutrient media, depending on the substrate or carbon source, the Penicillium occitanis Pol6 strain forms a carbohydrase complex containing cellulases, xylanases, pectinases, mannanases, β-glucosidase, laminarinases with activities 4.8–5.7 (FPA); 175-258; 52.3; 29.0; 17.0; 139.0 units / ml, respectively.

The problem to which the invention is directed, is the expansion of the arsenal of highly productive producer strains of the multienzyme complex containing a wide range of carbohydrases.

The technical result that can be obtained by carrying out the invention is to obtain a new producer strain of the complex with a higher level of activity of both cellulases and other complex components — β-glucanases, xylanases, pectinases and mannanases.

The essence of the invention is a new specially selected strain of mycelial fungus Penicillium funiculosum BKM F-3668 D producer of a complex of carbohydrases containing cellulases, β-glucanase, xylanase, pectinase and mannanase.

The strain of mycelial fungus Penicillium funiculosum is 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-3668D.

The strain is obtained using multistage classical mutagenesis and selection from the original culture of Penicillium funiculosum P1 (VKM F-3661D). The spore suspension of the original strain is irradiated with ultraviolet light. Irradiated spores are plated on Petri dishes with selective media, which are based on Hetchinson medium supplemented with 0.5% carboxymethyl cellulose (CMC), 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 manananases when cultured in a liquid medium in flasks. The most active variants selected during cultivation in flasks are again (repeatedly) subjected to irradiation and selection 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.

With growth on Maltz Agar (25 ° C), the diameter of the colonies reaches 36 mm on the 7th day of growth. The colonies are dense, even with a convex center. The mycelium is light, fluffy. The reverse side is fawn-reddish-orange. Conidiogenesis is weak, gray-greenish. Colonies on agar Chapek with yeast extract on the 7th day are 27-30 mm in diameter, folded, with a smooth edge, the surface is woolly. The mycelium is light, yellowish. The reverse side is fawn-orange. Conidiogenesis is weak, light, yellowish. Reverse - the side is fawn-orange. Conidiogenesis is weak.

The fungus does not grow on medium with glycerin and on апapek agar at 5 ° C. When cultivated on Chapek agar at 37 ° C, the colonies are 18 mm in diameter, folded, medium density, the reverse side is brownish-reddish.

Conidiogenesis: biverthicillate brushes, pressed metulae, smooth (8-10 × 2.5-3.0). Acerous phialides (8-9 × 2.5-3.0), with a short neck. Conidia are elliptical, small (2.2 × 1.5-2.0), smooth.

When cultured in deep conditions using soluble substrates (glucose, fructose, lactose), a loose branched mycelium with weak pelletization is formed, the specific initial growth rate of mycelium is 0.35 h ^-1 , at the end of cultivation 0.1 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 there is no very weak formation of cellulases and other carbohydrases. Nystatin resistance is good. With surface cultivation, it is resistant to a concentration of up to 0.5 μg / ml; at a concentration of 2.5 μg / ml, growth is inhibited. When digitonin (3.5-4.0 μg / ml) or Bengal pink (30-50 μg / ml) is added to the medium, the colony size decreases.

It is a prototroph. It is able to quickly assimilate glucose, lactose, glycerin, galactose, xylose, D-mannose, D-mannitol, trehalose, sorbose and sorbitol, slower - D-xylose, L- and D-arabinose, L-rhamnose and ribose. Weakly assimilates: D-glucosamine, deoxyribose, deoxygalactose, 2-deoxy-D-glucose and 5-thio-D-glucose.

Uses inorganic and organic nitrogen, assimilates the nitrate and ammonium forms of nitrogen well.

It forms enzyme systems that allow it to grow on the corresponding complex substrates: cellulose, starch, xylan, laminarin, β-glucan, lichenin, pectin, and galactomannan. Able to utilize lactic acid at a concentration below inhibitory.

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, amorphous cellulose, as well as the test repressor or antimetabolite (glucose, 2-deoxy-D-glucose, lactose, glycerin, etc. .). 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 destruction zone of amorphous cellulose (by the size of the clarification zone of the agar column in vitro) in the presence of a repressor or antimetabolite.

The obtained mutant Penicillium funiculosum No. VKM F-3668D, according to its morphological characteristics when growing on glucose potato agar, on sporulation agar (CM agar), differs from the original Penicillium funiculosum P1 strain by significantly reduced sporulation intensity, the color of the spore pigment (color) and the color of the spores is gray and colorless on the reverse side, when grown on agarized media, slower growth on solid media, increased ability, under deep cultivation on liquid media, to the biosynthesis of cellulases and carotene other than cellulases bogidraz β-glucanases, xylanases, pectinases and mannanases.

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 Penicillium funiculosum BKM F-3668D is carried out under aerobic conditions in a submerged state on a nutrient medium containing one or more substrates of carbon sources that are inducers of enzyme biosynthesis. As substrates can be used and substrates that are not directly inductors. The strain is capable of secreting a complex of enzymes - carbohydrases (cellulases, β-glucanases, xylanases, pectinases and mannanases) into the culture medium under appropriate conditions of the cultivation process based on the use of insoluble (MCC) and soluble (glucose) substrates. Glucose in the cultivation medium can be replaced by a cheaper product - starch hydrolyzate.

Cellulase activity is determined by FPA (according to the recommendation of SRAS), the activity of β-glucanases, xylanases, pectinases and mannanases in the culture fluid is determined by the ability to cleave β-glucan, xylan, polygalacturonic acid and galactomannan, respectively. The unit of activity is taken as such an amount of enzymes that within 1 min at a temperature of 50 ° C and pH 5.0 releases 1 μmol of reducing sugars equivalent to 1 μmol of glucose and determined by the Somogy-Nelson method (A.P. Sinitsyn, A.V. Gusakov, IM 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 preparations obtained by ultrafiltration or in the form of dry preparations.

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. To obtain seed (inoculum), the culture of the fungus Penicillium funiculosum BKM F-3668D is grown on wort or CM agar at 29 ° C for 7 days and then at room temperature in the light for 7 days. Sowing flasks is carried out 1 ml of a suspension of spores washed with agar with water containing 0.1% tween 80. Cultivation of the strain is carried out under aerobic conditions in 750 ml Erlenmeyer rocking flasks containing 100 ml of a liquid medium of the following composition, in g / l: glucose - 20.0; MCC - 20.0; corn extract - 10; (NHO ₂ SO ₄ - 5.0; KH ₂ PO ₄ - 3.0; MgSO ₄ × 7H ₂ O - 0.3; pH 4.5. The flasks are incubated on a shaker at 30 ° C and 200 rpm, in 144 hours

The activity of cellulases, β-glucanases, xylanases, pectinases and mannanases is 12.0; 150.0; 180.0; 10.0 and 8.0 units / ml, respectively.

Example 2. 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: microcrystalline cellulose (MCC) - 40.0; wheat bran - 10.0; yeast extract - 10.0; (NH ₄ ) ₂ SO ₄ - 5.0; KH ₂ PO ₄ - 5.0; MgSO ₄ × 7H ₂ O - 0.3; CaCl ₂ × 2H ₂ O - 0.3; pH 4.5.

The activity of cellulases, β-glucanases, xylanases, pectinases and mannanases for 18 hours of cultivation is 18.0; 310.0; 420.0; 20.0 and 15.0 units / ml, respectively.

Example 3. A cultivation process is carried out in an ANKUM 2M type fermenter with a working volume of 6.0 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 36 hours of cultivation on Erlenmeyer rocking flasks containing a liquid nutrient medium of the following composition, in g / l: glucose - 14.0; yeast extract - 10.0; KH ₂ PO ₄ - 10.0; MgSO ₄ × 7H ₂ O - 0.3; CaCl ₂ 0.2; (NH ₄ ) SO ₄ - 5.0; pH 4.5.

The first phase of cultivation (in which the fungus mainly grows and accumulates biomass) is carried out for 24 hours at 32 ° C on a liquid nutrient medium having the composition as described in example 2. After 24 hours, the second phase of cultivation begins (during which the accumulation of enzymes in a growth environment). In the second phase, glucose is continuously added to the fermenter so that its concentration in the medium does not exceed 0.5 g / L. The temperature in the second phase is maintained at 28 ° C and pH 4.0. Fermentation ends after 144 hours, by the end of fermentation, the initial volume of medium in the fermenter increases due to the introduced glucose solution by 20% and amounts to 7-7.5 liters.

The activities of cellulases, β-glucanases, xylanases, pectinases and mannanases for 124-144 hours of cultivation 30.0; 550.0; 630.0; 38.0 and 32.0 u / ml, respectively.

Example 4. The process is carried out in a fermenter type ANKUM 2M. The fermentation mode is the same as in example 3. The cultivation is carried out on the medium of the composition shown in example 3, but the concentration of glucose and cellulose in the first phase of cultivation in the composition of the liquid nutrient medium is increased to 40 and 60 g / l, respectively. In the second phase of cultivation, glucose is added in the same way as in Example 3. In the fermentation process, 60 hours after its start, mineral salts are added to the medium in an amount that was added at the beginning of the cultivation process, and after 60 and 96 hours, MCC in an amount so that its concentration was 2 g / l of fermentation medium.

After 144 hours of fermentation, the activity of cellulases, β-glucanases, xylanases, pectinases and mannanases is 35.0; 820.0; 990.0; 50.0 and 37.0 units / ml, respectively.

Thus, the proposed Penicillium funiculosum VKM F 3668D strain has the ability to produce a complex of highly active carbohydrases, including cellulases, β-glucanases, xylanases, pectinases and mannanases, which makes it possible to obtain a much more active and complete complex of enzymes, 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 Penicillium funiculosum BKM F-3668D (All-Russian Collection of Microorganisms at the IBPM named after G.K.Skryabin RAS) is a producer of a complex of carbohydrases containing cellulases, β-glucanase, xylanase, pectinase and mannanase.