RU2195490C2 - Strain of mycelioid fungus trichoderma longibrachiatum as producer of carbohydrases complex containing cellulase, beta-glucanase, xylanase, pectinase and mannanase - Google Patents

Abstract

FIELD: biotechnology, mycology, biochemistry, enzymology. SUBSTANCE: strain of mycelioid fungus Trichoderma longibrachiatum TW-1 VKM F-3634D is obtained by method of multiple classical mutagenesis and selection from the parent culture Trichoderma reesei QM 6a using ultraviolet irradiation. Strain shows ability to produce complex of highly active carbohydrases that makes a possibility of full complex of enzymes and also if necessary individual enzymes (components) of complex. For achievement of high productivity of the strain the use of complex and expensive nutrient media is not required. Nutrient media conventionally using in industrial technologies for preparing similar enzyme preparations are used for culturing. Invention can be used in food industry and agriculture. Invention allows to expanse effectiveness and to broad spectrum of enzyme preparations using in different fields of biotechnology and, especially, as fodder additions. EFFECT: valuable properties of fungus strain. 5 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 beta-glucanolytic, cellulose, hemicellulolytic and pectolytic enzymes that lyse 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 biodegradation as well as in the food and alcohol industries, in brewing, as additives in feed, for silage of feed in agriculture.

 Many strains are known that produce individual carbohydrases, mainly cellulases, and methods for their cultivation under aerobic conditions.

 As a rule, microorganisms, in particular mycelial fungi, form a complex of enzymes that destroy plant substrates. Usually, the complex includes mainly cellulases, as well as other carbohydrases, among which xylanases 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, beta-glucosidase, beta-xylosidase, beta-glucanase and laminarinase (RF Patent 2057179, CL 12 N 9/42, 1996). The activity of enzymes in the culture fluid are respectively: cellulase - 5.0 U / ml; xylanase - 125.0 U / ml; beta-glucosidase - 20.0 U / ml; beta-xylosidase - 36.0 U / ml; beta-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.

 As is known, the most active producers of cellulases are strains of mycelial fungi of the genus Trichoderma. In order to increase their ability to produce and adapt to cheaper environments, various mutant and recombinant strains of Trichoderma longibrachiatum (syn. Trichoderma reesei) were obtained. The mutant Trichoderma.reesei MCG 80, when continuously cultured on a nutrient medium with 8% cellulose and biotin, provides a cellulase activity of 17.2 u / ml FPA (US Patent 4,472,504, class C 12 N 9/42, 1984). FPA is the activity of the cellulase complex, determined by filter paper and expressed in international units according to the IUPAC recommendation (T.K. Chose., Pure and Appl. Chem., Vol. 59, 2, pp. 257-268).

 Known mutant Trichoderma.reesei IMET 43803, which due to a partial decrease in the catabolite repression of biosynthesis provides high cellulase productivity and high (up to 10 units / ml FPA) cellulase activity when cultured on a medium with lactose (patent GDR 291673, class C 12 N 9 / 42, 1991).

 Closest to the technical nature of the present invention is the mutant Trichoderma.reesei BCM 18.2 / QC (VGNKI-28), obtained by parasexual processes from the original culture of Trichoderma reesei IMET 43803, which has increased productivity and allows for high-activity enzymes in single-stage fermentation ( cellulase) per unit mass of the substrate used (RF Patent 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 in 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), cultivation in a fermenter on whey -12-14 u / ml (100-110 h).

 However, as follows from the information obtained at our disposal as a result of scientific research, fungi of the genus Trichoderma, as a rule, have high productivity, mainly in relation to cellulases, but to a much lesser extent in relation to other carbohydrases (beta-glucanases, xylanases, pectinases, mannanases), necessary for the hydrolysis of various polysaccharides included (in addition to cellulose) in the composition of plant materials. The strain Trichoderma reesei BCM 18.2 / KK (VGNKI-28) is not an exception in this case and has the same drawback as other Trichodrema strains, namely, it has low productivity for carbohydrases that are not cellulases (beta-glucanases, xylanases, pectinases , mannanases).

 The problem to which the invention is directed, is to obtain a new highly active strain of mycelial fungi - producer of a multienzyme complex containing a wide range of carbohydrases and suitable for cultivation on cheap raw materials in the presence of high concentrations of sugars.

 The technical result that can be obtained by carrying out the invention is to provide a high level of activity of both cellulases and other components of the complex - beta-glucanases, xylanases and pectinases in a fermentation medium, as well as to increase the ability to produce mannanase.

 The essence of the object of the invention is a new specially selected strain of mycelial fungus Trichoderma longibrachiatum TW-1 - producer of a complex of carbohydrases containing cellulases, beta-glucanase, xylanase, pectinase and mannanase.

 The strain of mycelial fungus Trichoderma longibrachiatum TW-1 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 VKM F-3634 D.

The strain is obtained using multistage classical mutagenesis and selection from the original culture of Trichoderma reesei QM 6a (VKM F-2047). The spore suspension of the original strain is irradiated with ultraviolet light. Irradiated spores are plated on Petri dishes with selective media, the basis of which is 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, beta-glucanases, xylanases, pectinases and mannanases 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 ^o С with obligatory reseeding at least once for 3-6 months.

Cultural and morphological characteristics of the strain
With growth on Maltz-agar, the diameter of the colonies reaches 80-90 mm on the 7th day, with growth at 25 ^o C. Mycelium develops mainly substrate, hyaline; airy meager, white. The conidial zone is formed by numerous compact pads with a diameter of up to 2 mm, originally white in color, green with age. The reverse side is light greenish yellow. Conidiophores are unpainted, smooth-walled, formed mainly on the substrate mycelium, the main branches are long and straight, sometimes crimped, up to 5 mm wide at the base, gradually tapering to the apex to 2 microns wide; lateral branches form at irregular intervals, usually short, are formed at an angle to the apex of the conidiophores. The phialides are single, club-shaped or broadly bottle-shaped, often curved, size - 5-10 h 2-3 microns. Conidia are unicellular, greenish, smooth-walled, ellipsoidal, 4-7 hours 2.5-4 microns, collected in small heads at the tops of the phialids.

When cultured 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 0.3 h ^-1 , at the end of cultivation - 0.1 h ^-1 .

Physiological and biochemical characteristics of the strain
Mesophilen. The optimum growth temperature of the mycelium is 32 ^o C (29-34 ^o C), the optimum for the formation of cellulases is 28 ^o C (26-29 ^o 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 a very weak formation of cellulases and other carbohydrases.

 Nystatin resistance is weak. 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 completely inhibited. With the addition of digitonin (3.5-4.0 μg / ml) or Bengal pink (30-50 μg / ml) to the medium, the colony size decreases.

 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-glucosamia, deoxyribose, deoxygalactose, 2-deoxy-D-glucose, 5-thio-D-glucose.

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

 It forms enzyme systems that allow it to grow on the corresponding complex substrates: cellulose, starch, xylan, laminarin, beta-glucan, lichenin, pectin, galactomannan and chitin. 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 carbohydrases 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 the tube) in the presence of a repressor or antimetabolite).

 The obtained mutant Trichoderma longibrachiatum BKM F-3634D in its morphological characteristics when growing on glucose-potato agar, on sporulation agar (CM agar) differs from the original Trichoderma reesei QM 6a strain by significantly reduced sporulation intensity, color of spore pigment and color of spore pigment and greenish yellow) on the reverse side, when grown on agarized media, a slower growth on solid media, an increased ability to deep cellulose biosynthesis of liquid media and other than cellulase, carb hydrazo - beta-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 Trichoderma longibrachiatum BKM F-3634D 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 may also be used as substrates. The strain is capable of secreting a complex of enzymes - carbohydrases (cellulase, beta-glucanases, xylanases, pectinases, mannanases) into the culture medium under appropriate conditions of the cultivation process based on the use of soluble substrates, for example glucose or lactose. 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 URAC), the activity of beta-glucanases, xylanases, pectinases and mannanases in the culture fluid is determined by the ability to cleave beta-glucan, xylan, polygalacturonic acid and galactomannan, respectively. For the unit of activity, take such an amount of enzymes that for 1 min at a temperature of 50 ^o C and pH 5.0 it 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 that do not limit the scope and essence of the claims associated with them.

Example 1. To obtain seed (inoculum), the culture of the fungus Trichoderma longibrachiatum BKM F-3634D is grown on wort or CM agar at 29 ^o C for 7 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. 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: beet pulp - 40.0; malt sprouts - 14.0; (NH ₄ ) ₂ SO ₄ - 6.0; KH ₂ RO ₄ - 2.0; MgSO ₄ • 7H ₂ O - 0.6; pH 5.4. The flasks are incubated on a shaker at 30 ^o C and 200 rpm for 120 hours

 The activity of cellulases, beta-glucanases, xylanases, pectinases and mannanases is 6.5; 25.0; 20.0; 7.0 and 0.5 u / 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) - 4.0; lactose - 20.0; (NH ₄ ) ₂ SO ₄ - 6.0; KH ₂ PO ₄ - 2.0; MgSO ₄ • 7H ₂ O - 0.6; pH 5.4.

 The activity of cellulases, beta-glucanases, xylanases, pectinases and mannanases is 6.0 hours at 120 hours of cultivation; 30.2; 22.0; 7.5 and 1.2 u / 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 (as described in example 1).

The first phase of cultivation (in which the fungus mainly grows and accumulates biomass) is carried out for 24 hours at 32 ^o C on a liquid nutrient medium of the following composition, in g / l: MCC - 4.0; lactose - 20.0; (NH ₄ ) ₂ SO ₄ - 6.0; KH ₂ RO ₄ - 2.0; MgSO ₄ • 7H ₂ O - 0.6. After 24 hours, the second phase of cultivation begins (during which the accumulation of enzymes in the growth medium). In the second phase, lactose is continuously added to the fermenter so that its concentration in the medium does not exceed the level of 1-2 g / l. The temperature in the second phase is maintained at 28 ^{° C.} , and the pH is 4.2. Fermentation ends after 120 hours, by the end of fermentation, the initial volume of the medium in the fermenter increases due to the introduced lactose solution by 50% and amounts to 9-9.5 liters.

 The activity of cellulases, beta-glucanases, xylanases, pectinases and mannanases for 110-120 hours of cultivation of 20.0; 380.0; 65.0; 23.0 and 24.0 units / 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 a medium of the composition shown in example 3, but instead of lactose in the first phase of cultivation, an enzymatic starch hydrolyzate is used in the liquid nutrient medium (600 ml per 6.5 liter fermenter, initial glucose concentration in the environment 2.9-3.3%), and instead of MCC - wheat bran at a concentration of 40 g / l. In the second phase of cultivation, lactose is added in the same manner as in Example 3. In the fermentation process, 60 hours after its start, mineral salts are added to the medium in the amount that was added at the beginning of the cultivation process.

 After 120 hours of fermentation, the activity of cellulases, beta-glucanases, xylanases, pectinases and mannanases is 21.0; 470.0; 90.0; 35.0 and 30.0 units / ml, respectively.

 Example 5. 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 4, but in addition to wheat bran and starch hydrolyzate in the composition of the liquid nutrient medium, MCC at a concentration of 5 g / l is also used in the first phase of cultivation. In the second phase of cultivation, lactose is added in the same way as in Example 3. In the fermentation process, 40 and 84 hours after its start, MCC is added to the medium in an amount so that its concentration is 2 g / l of the fermentation medium, and after 60 hours - mineral salts in the amount that was added at the beginning of the first phase of the cultivation process.

 After 120 hours of fermentation, the activity of cellulases, beta-glucanases, xylanases, pectinases and mannanases is 25.0; 510.0; 108.0; 40.0 and 35.0 units / ml, respectively.

 Thus, the proposed strain Trichoderma longibrachiatum VKM F-3634D has the ability to produce a complex of highly active carbohydrases, including cellulases, xylanases, pectinases, beta-glucanases and mannanases, which makes it possible to obtain a 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 Trichoderma longibrachiatum VKM F-3634 D (All-Russian Collection of Microorganisms at the IBPM named after G.K. Scriabin RAS) is a producer of a complex of carbohydrases containing cellulases, beta-glucanase, xylanase, pectinase and mannanase.