CS261056B1 - Strain of microorganism trichoderma reesei ccm-f-806 - Google Patents

Abstract

The solution concerns a new Trichoderma fungus strain reesei F-806, for which the creation is cellulolytic enzymes partially resistant to catabolic repression easily utilizable sugar substrates. The essence The solution is to have the strain described prepared by mutagenesis in a known manner from the Trichoderma reesei F-560 strain it does not show resistance to catabolic repression. New Trichoderma reesei F- -806 can be used in industrial production! cellulolytic enzymes.

Description

The present invention relates to a new strain of imperfect microscopic fungus Trichoderma reesei CCII producing increased amounts of cellulolytic enzymes in submerged culture, wherein the production of cellulases is partially resistant to catabolic repression by readily utilizable sugars.

Microscopic filamentous fungi of the genus Trichoderma are good producers of cellulolytic enzymes (cellulases) when powdered cellulose or spirits containing cellulose are used as both substrate and inducer. The maximum cellulase activity achieved with cellulose as substrate varies from 70 to 200 nkat / ml FPA in batch culture. W. Khan, K. A. Lamb, Biotechnol. Lett. 6, 633 (1984); Watson T. G., I. Nelligan, 5, 25 (1983); H. Matanič et al. Prehramb. Tecbnol. Rev. 18, 3 (1980)]. However, the disadvantage of using cellulose and other water-insoluble cellulose substrates is that the production microorganisms in such growth media and produce cellulose relatively slowly - one production cycle in batch submerged culture takes 10-14 days. Another disadvantage of using insoluble substrates is that by their presence they increase the viscosity of the nutrient medium, which increases the energy requirements for mixing and impairs oxygen transfer in the culture mixture. In addition, the use of insoluble cellulosic substrates complicates the way they are metered in the course of a continuous or continuous process. fed-batch cultivation. Use of soluble carbon-glucose sources, respectively. However, due to catabolic repression, cellulase production is strongly suppressed. Therefore, such strains of producers are sought in which catabolic repression of cellulase formation is removed by mutation. The rate of removal of catabolic repression is determined by the maximum concentration of sugar (glucose, lactose, etc.) at which the fungus is still capable of producing cellulose.

These drawbacks are overcome by the present invention, which is a new strain of Trichoderma reesei, a CCII mutant deposited in Cs. Collection of Microorganisms of the University of J. E. Purkyně in Brno, Class Defender of Peace 10 under the designation CCM F-806. The new strain exhibits increased cellulase productivity and has partially mutated catabolic repression of cellulase formation when grown on easily utilizable carbohydrate substrates.

Said new strain was obtained by a known method, a combined physical and chemical mutagenase associated with a two-step enrichment of mutagenized spores according to I. Labudova and V. Farkas FEMS Microbiol. Lett. 20, 211 (1983) from the starting Trichoderma reesei strain CCM F-560.

The main advantage of the new strain compared to the original strain is an approximately 8-fold increase in cellulase production when cultivated on 1% cellulose as a substrate where activity is achieved depending on the cultivation conditions

FPA 50-180 nkat / ml on day 5 of culture. Another advantage of the new strain is the partial elimination of catabolic repression of cellulase formation, which is manifested by its ability to produce celluloses even in the presence of 0.05% glucose, respectively. 0.2% lactose in the medium, while the original T. reesei F-560 strain produced no cellulose under these conditions.

Example 1

Description of the new tribe

Morphology: fibrous, hutso-branched, rarely septic when grown on agar glucose medium. Diameters of 3,5 to 5 μΐη. Condicids shorter and branched, violet usually single or opposite. Conidia abundant, initially white, then lived in a ripened state of green to green-brown pigmented, elliptic-ovoid, diameter 2 to 3 µm. Conidiation occurs in submerged cultivation with good aeration and in a medium that is low in carbon and nitrogen. When cultivated on a solid surface, conidation is stimulated by light. Submerged cultivation sometimes produces spherical chlamydospores with a diameter of 8 to 10 μΐη. When cultivating on glucose, respectively. fructose secretes a lived pigment into the medium. It differs morphologically in comparison with the flood strain by the higher branching density of the mycelium and by 50 to 100% / o larger thick hyphae.

The microorganism grows on a variety of sugar substrates: cellulose, xylane, D-glucose, D-galactose, D-mannose, D-fructose, D-xylose, L-arabinose, D-ribose, cellobiose, trehalose, starch, iaminarin, maltose, D-glycerol, ethanol. It grows poorly on: sucrose, raffinose, acetate, lactate, tartrate, malonate, inulin and chitin. The optimum growth temperature is 28-32 ° C, optimally pH 3-5, does not grow at 50 ° C.

Example 2

Preparation of a new tribe

Conidia Trichoderma reesei OM 6a is suspended! at a concentration of 10 ⁸ / ml in 10 ml of a 0.05 mol / l solution of Tris (hydroxymethyl) aminomethane-hydrochloric acid (Tris-HCl) pH 9.6, containing 0.02 why, polyoxyethylene sorbitan tristeic acid (Tween 80) and 0, 05% N-methyl-N'-nitro-N-nitrosoguanidine and the suspension is irradiated on a Petri dish under a 40 W UV lamp from a distance of 20 cm for 5 min. At the end of the irradiation, the suspension is incubated for 6 hours at 37 ^° C. they are then inoculated into 40 ml of minimal medium containing 5 g of ammonium sulfate, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium heptahydrate, a mixture of vitamins according to LJ Wickereham [US Dept. Agric. Bul. No. 1029, 56 (1951) )],

In addition, 2% cellobiose and 0.5% 2-deoxy-D-glucose are added to the medium. The mixture was incubated on a rotary shaker at 30 ° C for 4 to 5 days until the first germinating conidia appeared. These are then separated from non-germinated spores by filtration through a S1 glass frit and sown on sporulation agar containing at least medium plus 2% glucose and allowed to grow in the light at room temperature for 5 days. The conidia formed are washed off the surface of the agar with sterile water and inoculated into minimal medium containing 0.5% Na-carboxymethylcellulose and 0.5% 2-deoxy-D-glucose and incubated for several days at 30 ° C on a shaker. The germinated conidia are separated by filtration through a S1 frit and sown on detection agar containing at least medium plus 0.5 to 1% amorphous cellulose, 5% by weight of fructose, respectively. glucose or glycerol and 50 µg / ml tetrajodotetrachlorofluorescein disodium salt (Bengal red.) Selection agar plates are incubated for 2 days at 30 ° C and 1 day at 50 ° C. The isolated colonies are purified by repeated conidization and passage, and individual clones are tested for their ability to produce cellulolytic enzymes in the presence of cellulose and lactose as substrates.

Example 3

The medium containing 1 g of 20 g lactose, 1.5 g of potassium dihydrogen phosphate, 5 g of ammonium sulfate, 0.3 g of magnesium sulfate, 0.2 g of calcium chloride and 2.5 g of yeast autolysate in water is inoculated with 10 ⁵ conidia / ml of Trichoderma reesei CCII and cultured at pH 4.0-5.0 in a fermentation tank at 30 ° C, stirring at 6 Hz and aeration. 1 volume (s) min for 5-7 days. During fermentation, cellulose FPA activity of 60-80 ncatal / ml is obtained in the medium, determined according to the IUPAC Recommendation (Measurement of Cellulase Activities, Commision on Biotechnology, IUPAC. New Delhi, 1985) ).

The Trichoderma reesei F-806 strain can be used in the industrial production of cellulases on various carbohydrate substrates, preferably on substrates containing readily utilizable carbohydrates.

Claims (1)

In addition, 2% whole-biose and 0.5% 2-deoxy-D-glucose are added to the medium. The mixture is incubated on a rotary shaker at 30 ° C for 4-5 days until the first germination conidia appears. These were then separated from the non-germinated spores by filtration through a S1 glass frit and sown on a sporulatag containing at least a plus 2% glucose medium and allowed to grow at room temperature for 5 days. Generated conidia are washed off the agar surface with sterile water and seeded into a minimal medium containing 0.5% Na-carboxymethylcellulose and 0.5% 2-deoxy-D-glucose and incubated for several days at 30 ° C on a trek. The germinated conidia are separated by filtration through a S1 frit and sown on the detection agar containing at least mediumplus 0.5-1% amorphous cellulose, 5% fructose, respectively. glucose, or glycerol and 50 µg / ml of tetra iodotetrachlorofluorescein disodium salt (Bengal red. Selective agar plates are incubated for 2 days at 30 ° C and 1 day at 50 ° C. Colonies isolated on selective agar) The purified colonies are purified by repetitive coagulation and passage, and the individual clones 6 are tested for their ability to produce cellulose enzymes in the presence of cellulose and lactose as substrates. lactose, 1.5 g of potassium dihydrogen phosphate, 5 g of ammonium sulfate, 0.3 g of magnesium sulfate, 0.2 g of calcium chloride and 2.5 g of fermented autolysate in water, inoculated with 105 conidia / ml of Trichoderma reesei CCIIa cultured at pH 4.0 to 5.0 in a fermentation tank at 30 ° C, stirring 6 Hza aeration 1 volume (volume) min form 5 to 7 days, During fermentation, the cellulose activity of FPA60 to 80 is obtained in the medium. natal / ml, as determined by IUPAC (Measurement of Cellulaseactivities, Commision on Biotechnology, IUPAC). New Delhi, 1985). The Trichoderma reesei F-806 strain can be utilized in the industrial production of cellulase-born carbohydrate substrates, preferably on substrates containing fat-soluble carbohydrates. OBJECT OF THE FITTING The strain of Trichoderma reesei F-806 with partially catabolically derepressed cellulase production.