The culture medium to obtain the enzyme cellulase in its industrial production by the method of deep cultivation of the fungus trichoderma viride 44-11-62/3 and the method of producing the enzyme cellulase in this environment

 

(57) Abstract:

The invention relates to biotechnology, to the biosynthesis of cellulase. The method involves deep cultivation of the fungus Trichoderma viride 44-11-62/3 in a medium containing as a carbon source hydrolysates of corn starch and wheat bran. The fermentation process is carried out at discrete introducing an activator solution of lactose to 0.5%. The use of homogeneous nutrient medium allows to exclude mechanical losses in production. The method achieves higher cellulase activity in the culture fluid 27 u/ml, increasing the yield of the manufacturing process enzyme preparation of cellovyridin by 24%. 2 S. p. f-crystals.

The invention relates to the field of biotechnology, and more precisely to its initial stage - the biosynthesis of enzymes, which are produced in the culture fluid during submerged cultivation of the lower fungi on artificial media.

Such enzyme preparations include cellulose-containing substances that occur in the culture fluid during submerged cultivation of the lower of the fungus Trichoderma viride. This enzyme further receive technical enzyme preparation cellularity feed from trudnousvoyaemoe animals and poultry grain (rye, barley, oats, etc ).

The initial stage of obtaining celloviridin GSH is deep lower cultivation of the fungus Trichoderma viride in a fermentation apparatus. Then, the obtained culture liquid is passed through pipelines to the spray dryer type FEC-100, where, after its drying obtain the target product.

Success produce a marketable product largely depends on the composition of the nutrient medium on which is cultivated Trichoderma viride, and fermentation conditions. How deep cultivation of mushroom producers of Trichoderma and composition of nutrient media have been well studied at the laboratory level. For example, in the work of C. A. Velichko, T. C. Polansky and other "Method of preparation of nutrient media for culturing producers of cellulolyticus enzymes". USSR author's certificate N 3486197/28-13, CL C 12 N 9/42, C 12 N 1/22 published 30.01.84. The authors use for the cultivation of Trichoderma viride Trichoderma long. , Egectridum candidium and other nutrient medium of the following composition, %:

Beet pulp - 3

Ammonium nitrate - 0,2

Potassium dihydrophosphate - 0,2

Malt sprouts - 0,5

Wheat bran - 0,2

PH environment set to 4.5 pH. Pre-mix cellulocutaneous the celloviridin GSH or callosomarginal HRP in amount of 0.2-2.0 and 0.1 to 1.0 wt.% at a temperature of 40-55oC for one hour, the water ratio of 1.5 to 1:10. The output of cellulase after cultivation of the seven days in a 10-liter fermenter was 12.2 IU/ml

In the work of C. P. Salovarova, Y. P. Grachev and M. S. Vaganova "Influence of nutrient medium composition on the biosynthesis of cellulolyticus enzymes in submerged cultivation of the fungus Trichoderma long. 7-26" Applied biochemistry and Microbiology, 1978, T. 14, vol.2, S. 172-176 methods of mathematical modeling, confirmed the experimental data, the authors showed that the optimum environment for submerged cultivation of mushroom culture Trichoderma is a medium of the following composition, %:

Beet pulp - 4,6

Ammonium nitrate - 0,7

Potassium dihydrophosphate - 0,3

Malt sprouts and wheat bran 1:1 - 0,75

The specific enzymatic activity of cellulase under cultivation in 750 ml flasks for Trichoderma long/ 7-26 is 16,3 u/ml Fermentation spend seven days in continuous mode at a temperature of (30 1)oC. At the end of fermentation, the pH value of the culture fluid zakislyaetsya.

In this paper, the Method of obtaining cellulolyticus enzymes", N. T. Shinkarenko, N. A. ostrakova and others, USSR author's certificate N 3449898/28-13, CL C 12 N 9/42 published 15.02.84, in the 4% of chromium sulfate.

When the fungus cultivation in continuous mode for 108 h 60-liter fermentors received output of the enzyme with the activity of 15.5 u/ml

As can be seen from these works, a necessary condition for the cultivation of Trichoderma viride is the presence of a nutrient medium beet pulp as a carbon source.

Famous work "Method of production of cellulases" E. C. frost, L. I. Sazhin and others , USSR author's certificate N 4271847/31-13, CL C 12 N 9/42//(C 12 N 9/42, C 12 R 1:885), published 07.07.89, which describes the cultivation of Trichoderma viride strain F-120 and F-79 on 93the fermenters. While beet pulp authors replaced tsellolignin (at a ratio of cellulose and lignin 1: 1). It got deep hydrolysis enzyme preparations by allopurinol, allocation, celloviridin rate of 20 u/g of material within 48-72 hours at a temperature of 45-50oC with the subsequent laundering of the resulting sugars to a concentration of not less than 0,002%. When this got out of cellulase and 22.6 u/ml

The disadvantages of this method are the duration and complexity of the procedure of preparation of the nutrient medium.

In the "Strain of the fungus Trichoderma viride VKPM F-374 - producer of extracellular cellulase and xylanase", N. A. Ostrikov, S. A. Konovalov and dstanley conditions of its cultivation in 10 liter fermentors in a nutrient medium of the following composition, %:

Beet pulp - 3,0

Ammonium nitrate - 0,4

Potassium dihydrophosphate - 0,2

The ammonium sulfate - 0,4

Magnesium sulfate - 0,03

Malt sprouts - 1,43

Potato bard - 5,0

PH environment pH of 5.5. The cultivation is carried out in continuous mode seven days. Out of cellulase 35 u/ml

The production of cellulase by 63 m3the fermenters are described in the regulation on the cellovyridin GSH "ODA 015800805-66-96 for the production of drug cellovyridin GSH, JSC "Biotechnology", 1996". Source biosynthesis of cellulase here is the lowest fungus Trichoderma viride 44-11-62/3. Recommended regulatory environment for cultivation, %:

Beet pulp - 2,9

Malt sprouts - 1,4

Lactose - 0,3

Bolotin - 0,2

The ammonium sulfate - 0,7

Potassium dihydrophosphate - 1,0

Magnesium sulfate - 0,05

Cellulose - 0,9

PH environment pH of 5.5. The cultivation is carried out in continuous mode seven days. The specific activity of the enzyme in the culture fluid 30 u/ml Volume of culture fluid 32 m3when the load factor of 0.6.

This method of obtaining fungal culture of Trichoderma viride we accept for the prototype because it is closest to the claimed method.

3is that as carbon source in the nutrient medium used beet pulp. He even after crushing has a porous structure that swells in water and floats, which leads to the formation of abundant foam. This causes the formation of congestion in technological pipelines. For jam processing operation must be interrupted. In addition, this fact adversely affects the performance of the process equipment. All this leads to significant mechanical losses as a nutrient medium, and the culture fluid and, consequently, to low yield of the final product. The decline in output is due mainly low technology medium, associated with the inhomogeneity of its composition due to the use of sugar beet pulp as a carbon source. And finally, the specific activity of cellulase during playback of regulatory regimes on the proposed nutrient medium does not exceed 23 u/ml, which also leads to a reduction of yield in the production of celloviridin GSH.

The aim of the invention is to increase the output of the process for obtaining an enzyme preparation celloviridin when industrial production is ananti do not form blocks or foam. Well subjected to heat sterilization on the continuous sterilization - ONS-20. This will eliminate the causes of mechanical losses. This goal is achieved by the fact that the proposed method of producing cellulase from the fermentation medium as a carbon source instead of the sugar beet pulp is used corn starch and wheat bran, hydrolyzed to the available sugars enzyme preparations by glucosamina GSH and Miloserdiya GSH that gives it a new quality - homogeneity. Productivity biosynthesis of cellulase should be at least 20-25 IU/ml. This goal is achieved by the fact that the proposed method of producing cellulase from the fermentation medium as a carbon source instead of the sugar beet pulp is used corn starch and wheat bran, hydrolyzed to the available sugars enzyme preparations by glucosamina GSH and Miloserdiya GSH. When the fermentation process is carried out at discrete introducing an activator solution of lactose throughout the biosynthesis of cellulase in the culture fluid to 27 u/ml, or 15% above recommended specs.

The invention consists in the optimal ratio to the environment to obtain the enzyme cellulase, containing potassium dihydrophosphate, magnesium sulfate, cellulose, lactose and water, characterized in that it further comprises calcium chloride, ammonium phosphate, yeast and enzymatic hydrolysates of wheat bran and corn starch in the following ratio of components, %:

Enzymatic hydrolysate of corn starch and 1.5

Enzymatic hydrolysate of wheat bran - 3,0

Calcium chloride - 0,05

Phosphate ammonium - 0,6

Potassium dihydrophosphate - 0,3

Yeast - 0,15

Magnesium sulfate - 0,05

Cellulose - 0,5

Lactose - 0,5

Water tap - Rest

Based on 1 kg of starch add 3000 units of amylase activity and 6000 units of activity glucoamylase. 1 g of pulp add 12 units of activity of cellulase (separately prepare a feed solution of lactose at the rate of 0.5% for the entire fermentation process). A solution of lactose contribute discrete portions. The culture medium is homogeneous in its composition and has a high adaptability in the production of cellulase in industrial fermentor. Virtually no forms of congestion, which significantly reduces mechanical losses. Discrete dosage solution of lactose allows to achieve the desired production which leads to an increase in output for the industrial production of cellovyridin GSH by 24%. Process becomes more predictable and repeatable.

As examples consider the processes of cultivation lowest of the fungus Trichoderma viride 44-11 - 62/3.

Example 1 (the prototype)

Nutrient medium 63 m3the fermenter is prepared on the basis of the following composition, %:

Beet pulp - 2,9

Malt sprouts - 1,4

Lactose - 0,3

Bolotin - 0,2

The ammonium sulfate - 0,7

Potassium dihydrophosphate - 1,0

Magnesium sulfate - 0,05

Cellulose - 0,9

PH environment of 5.5 pH.

Separately prepare carbohydrate ingredients: sugar beet pulp, malt sprouts, cellulose, Bolotin separately and salt components: ammonium sulfate, lactose, potassium dihydrophosphate, magnesium sulfate. They are then sterilized with live steam in a continuous sterilization at a temperature 124-130oC alternately feeding into the mixer. From the mixer on technological lines ready environment served in the fermenter. Then make a seed lower fungus Trichoderma viride 44-11-62/3. Cultivation spend seven days at a temperature of (301)oC. Sterile air supplied, on the assumption of 0.3 volume/h per 1 volume of culture liquid of the first 12 h of culture growth, the offered pressure of 0.05 MPa. The rotation speed of the mixer 177 rpm Cultivation can be done without making aid in the fermentation process. On day 5-7 environment zakalilisy to the value of pH 3 pH units.

The obtained culture fluid having cellulase activity, technological pipelines transferred to the spray dryer FEC-100.

The estimated load factor of the fermenter to 0.6. Received 303the culture fluid with a specific enzymatic activity of the cellulase 23 IU/ml After drying the obtained 360 kg of cellovyridin GSH with enzymatic activity 330 u/, the Output of the process was 17.2%.

Example 2 (according to the claimed method)

A nutrient medium is prepared on the basis of the following composition, %:

Enzymatic hydrolysate of corn starch and 1.5

Enzymatic hydrolysate of wheat bran - 3,0

Calcium chloride - 0,05

Phosphate ammonium - 0,6

Potassium dihydrophosphate - 0,3

Yeast - 0,15

Magnesium sulfate - 0,05

Cellulose - 0,5

Lactose - 0,5

Water tap - Rest

Based on 1 kg of starch add 3000 units of amylase activity and 6000 units of activity glucoamylase. 1 g of pulp add 12 units of activity cellula the major components of the nutrient medium is prepared as follows. In the container of warm water (40-45oC) add the required amount of corn starch, wheat bran, calcium chloride and adjusted pH to a value of 6.0 to 6.5 pH with caustic soda solution. Download the estimated amount of amylase and spend the liquefaction of starch 3 hours Then the solution is cooled, adjusted the pH value to 4.5 to 4.7 pH solution of phosphoric acid and enter the estimated number of glucoamylase. The hydrolysis is carried out 1 h

In a separate bowl prepare the salt components. In hot water (40-45oC) add the estimated amount of the pulp, mix, bring the pH value to 4.5 to 4.7 pH solution of phosphoric acid, load the cellulase and at this temperature, carry out the hydrolysis of 2 hours Then make the rest of the salt components.

In the mixer salt and carbohydrate components are mixed and passed to the installation of continuous sterilization, where sterilized with live steam temperature 124-130oC.

The cultivation parameters analogous to example 1. But in the fermentation process after a certain time impose discrete portions of a solution of lactose, the basis of changes in the carbohydrate composition of the culture fluid.

Received xpecially dryer FEC-100. The estimated load factor of the fermenter to 0.6. Received 37 m culture liquid with a specific enzymatic activity of the cellulase 27 IU/ml After drying the obtained 920 kg celloviridin GSH with enzymatic activity 935 u/, the Output of the process was 41.3%.

Thus, the use of the proposed method, the output of the process of obtaining technical celloviridin GSH increased by 24.1%.

1. Nutrient medium to obtain the enzyme cellulase in its industrial production by the method of deep cultivation of the lower of the fungus Trichoderma viride 44-11-62/3 containing potassium dihydrophosphate, magnesium sulfate, cellulose, lactose and water water, characterized in that it further comprises calcium chloride, ammonium phosphate, yeast and enzymatic hydrolysates of wheat bran and corn starch in the following ratio of components, %:

Enzymatic hydrolysate of corn starch and 1.5

Enzymatic hydrolysate of wheat bran - 3,0

Calcium chloride - 0,05

Phosphate ammonium - 0,6

Potassium dihydrophosphate - 0,3

Yeast - 0,15

Magnesium sulfate - 0,05

Cellulose - 0,5

Lactose - 0,5

Water water Remained viride 44-11-62/3 nutrient medium and drying the culture fluid, characterized in that deep cultivation lead to a nutrient medium under item 1, with lactose contribute in the form of a solution of the discrete portions during the entire cultivation process.

 

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FIELD: biotechnology, in particular reagent for structural protein hydrolysis.

SUBSTANCE: method for production of protheolytic reagent includes cultivation of producer strain selected from dermatophyte of species Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton equinum, Microsporum canis, Microsporum gypseum, preferably from fungus strains capable to produce complex of structural protein proteinases (scleroproteases) with total protheolytic activity at least 0.7 U/mg including collagenase 0.1-4.5 U/mg; keratinase 0.1-0.5- U/mg; elastase 0.5-1.9 U/mg. Cultivation is carried out, for example, in wort agar-agar or in wort broth for 20-24 days.

EFFECT: scleroproteases with improved specific activity; method for protheolytic cleavage of specific substrates (scleroproteins) with increased rate and depth.

2 dwg, 12 ex

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