SU971877A1 - Medium for producing proteolytic enzymes - Google Patents

Description

The invention relates to the production of preparations of proteolytic enzymes and can be applied in the microbiological industry, biochemistry of micro organisms, medicine and hematology. Known medium for the production of ACUvrohi JCeS SplieVXjicles strains 35 proteases of caseinolitic and fibrinolytic action when acted upon. The medium contains glucose (6-7%), sodium citrate, (NH4) i504. KH2.PQ4- Sch InS04.TeS04 tl. The disadvantage is the complexity of the composition of the medium, the use of expensive and scarce compounds in it, some of them in large quantities, the duration of the inoculation of the seed and the insufficient yield of the desired product. Also known is the medium for the production of proteolytic enzymes by cultivating the producer AtiiMOmi ces rivflOSOS and microorganism stimulator of the AotinoTTWces vioEaceus torus, containing as a carbon source glkzo, as well as mineral salts (NH) 2 $ 0, CaCOj, Wig-S04 .FeSQ.Se. 2: i5O4 On this medium, the two AC-MtTOWscee rimosos producers, proteases and Ac-twon 5Ce9 vipEdCeus 5 -G inactive in relation to the synthesis of secreted proteases of the organism L 2 3, are cultured on-site, cultivating. The disadvantages of the known medium are the use in the medium of expensive products in large quantities (glucose 4%), the insufficiently high yield of the target product, the complexity of the medium composition and the duration of the growth of the seed material. The purpose of the invention is to simplify the composition of the medium, reduce its cost, and reduce the yield of proteases with increased specificity for fibrin. The goal is achieved by the fact that the medium for obtaining proteolytic

enzymes by cultivating the producer of Actinown ces of the microorganism Acti stimulant ICP ages viofcaceusS G, containing a source from a lerod and mineral salts, including calcium carbonate and disubstituted phosphoric acid potassium and water, according to the invention contains 1 to 10 as a carbon source as carbon , 2 and mineral salts ammonium chloride and sodium nitrate, in the following ratio, wt.%;

Glycerin1-1,2

Ammonium chloride O, OO2-p, OOZ Sodium nitrate0,002-0,02

Disubstituted phosphate

potassium 0,011-0,02

Calcium carbonate0, 3-O, 4

Water pipe

water the rest

Example. In the proposed environment g / l: glycerin - 11; NH4C6 - 0.02;

K2.HP04 - 0.2; NaNOs- 0.2; CaCOj-4, after sterilization, was introduced to cultivate a monoculture with AcViMoyiwcee riWOSUS 5% of a two-day liquid seed or for a mixed culture of 5% by volume of a two-day Ac-t.rimosus liquid seed and 1.25% by volume of a two-day Act.vio6aceu seed. 5G, grown on medium, g / l: soybean flour ZbU glucose - 40; KN2.P04 - 0.5; NqCe2, 5; CaCO- 6. Cultivation of a producer of proteases or a mixture of a producer with Act-iiriiomvces vioEaceos 5G is carried out deep, with flasks in 75O ml with ipo ml of medium in rocking chairs (220 rpm) for 9 hours at 28 ° C. At the end of this time, the Ac-biviOV VCe5 H T "O5ue culture fluid contains 2268 standard units / ml of fibrinolytic ac. 42 kaz. u / ml caseinolytic activity, and in a mixture - 4480 sr. u / ml u / ml of firinolytic activity and 68.4 kaz. units / ml caseinolytic activity. The desired product is obtained from the culture fluid after separation by centrifuging the cells with the method. salting out ammonium sulfate 0.8 saturation.

The obtained results show that on the proposed medium fibrinolytic and caseinolytic proteases are formed; the action for the protease component is 3 times more than in the known and for the mixture of actinomycetes 2.4 times. The specificity for fibrin in the proposed method increases by 2 times, while in the well-known it falls.

Example2. In the proposed media of the following composition, g / l; glycerin 10; MN4Se -0.02; BaMO, 5g 0.02; KiMPQ, 0, 11; 4 after sterilization, Aciinomvcee rinio5us5% monoculture of a two-day liquid seed or for mixed culture of 5% by volume of a two-day Actinowi ces vioEaceusS® mixed culture cultivated in medium, g / l; soy flour 30; glucose - 40; KNARO - O, 5; MASS 2, 5; CaCO - 6.0. The cultivation of a protease producer in a monoculture and a mixture of a producer with AcMviovvivces viotaceusS G is carried out as mentioned above. After 96 hours, the enzymatic activity is 2,073 srv. u / ml for fibrinolysis and 36.5 kaz. u / ml for caseinolysis. The desired product is obtained from the culture liquid after separation by centrifuging the cells by salting out ammonium sulfate at 0.6 saturation.

The proposed environment is more economical than the known one, the container does not contain scarce food components. The process of cultivation on the proposed medium takes less time than in the known one, since the cultivation of seed requires 2 days instead of 3 days in the known.

The table shows the advantages of the proposed environment.

From the data given in the table wine, that on the proposed medium in the monoculture of actinomycete, fibrinolytic activity is 3 times higher, and caseinolytic activity is 2 times higher than in the well-known. With mixed cultivation on a new medium, fibrinolysis increases 6 times compared with the pure culture of the known method, and caseinolysis only increases by 3.7 times, which leads to an increase in fibrin specificity by a factor of 2. Fibrin specificity is an important property of this kind of enzymes, as it provides preferential hydrolysis of fibrin without affecting blood proteins, which play a large role in hemostasis.

59718774

Thus, the invention implements the cost and increases the yield of proteases

allows you to simplify the composition of the medium, reducing the increased specificity for fibrin.

Claims (1)

CLAIMS medium for culturing proteolytic enzyme producing microorganism Actinomyces romosus AND-stimulant A ct inomy e se s viofcoc us 5G containing a carbon source and ^Mi-45 neralnye salts including dibasic potassium phosphate and water, calcium carbonate, otlichayuschayaGlitserin Ammonium Chloride Sodium Nitrate Potassium phosphate disubstituted Calcium carbonate tap water 1-1,2 0.002-0.003 0.002-0.02 0,011-0,02 0,3-0,4 The rest is that, in order to simplify the composition of the medium, reduce its cost and increase the yield of proteases with increased specificity for fibrin, the medium contains 1– 1.2% and mineral salts - ammonium chloride and sodium nitrate 55 in the following ratio, wt.%: VNIIPI Order 8479/9