UA120572C2 - BACTERUS STRENGTH BACILLUS SUBTILIS IMB B-7724 - PRODUCER OF CYTOTOXIC SUBSTANCES WITH ANTUCTUMOR ACTION - Google Patents

Abstract

The invention relates to a strain of Bacillus subtilis IMB B-7724, which is a producer of cytotoxic substances with antitumor activity, registered in the Depository of the Institute of Microbiology and Virology (IMB). D.K. Zabolotny National Academy of Sciences of Ukraine under the number IMB B-7724.

Description

The claimed useful model belongs to the field of microbiology and biotechnology, namely the cultivation of microorganisms - strains of Vasiyv5 5ybijv bacteria, and can be used to obtain biologically active substances of lectin and protease nature with cytotoxic activity against tumor cells.

Known strains of Vasipii5 z,iBiiv5 IMV 7025, Vasiiii5 5,iBii5 Z16M and Vasiiii5 royutuh 102 KDU, which produce biologically active substances of lectin and protease nature with antitumor activity (11. However, their productivity does not ensure the required level of the latter.

The closest strain to the one proposed is Vasiyi5 zibiyv IMV B-7025 |2I|, which produces biologically active substances of a lectin nature with cytotoxic antitumor activity against tumor cells of different histogenesis (sarcoma 37, adenocarcinoma

Ehrlich, MK/L lymphoma, OH-2 lymphosarcoma, OH-3 plasmacytoma, Lewis lung carcinoma).

Compared to the above-mentioned strains Vasiy5 5!ibBiiv IMV B-7025 produces a larger amount of lectin-type substance. However, its use is not always effective due to insufficiently high antitumor activity.

The basis of a useful model was the task of obtaining the Vasiinv5 5ibiyv strain, which is more effective and stable in the production of cytotoxic substances, in particular lectin and protease in nature, with high antitumor activity.

The task was solved by obtaining a new strain Vasiiysh5 v5yYibBiy5 using methods of analytical selection. The new strain was isolated from the Vasiya5 5!,ibBiiv 7 strain by selecting individual colonies with an increased ability to produce cytotoxic substances when cultivated under competitive conditions with the strains of microorganisms Yeieriosossiv ruodepie and Zbetaya tagsezsep5 and deposited in the collection of the Institute of Microbiology and Virology named after D.K. Zabolotnoi (MV) of the National Academy of Sciences of Ukraine under number IMV B-7724 (Certificate of initial deposit of the microorganism strain dated March 12, 2018). The strain was identified according to "Berga's Brief Identifier of Bacteria" IZ). As a result of the breeding work, this strain acquires such cultural-morphological and physiological-biochemical properties that ensure its ability to high productivity of cytotoxic substances with antitumor activity.

Cultural and morphological features. This strain of Vasiyya5 5!Yiriyv grows well at a temperature of 28-40 "C on meat-peptone broth (MPB), meat-peptone agar (MLA),

From Hause's semi-synthetic medium, 10 90 wheat bran extract. The microorganism is a gram-positive, rod-shaped cell with a width of 0.7-0.8 μm and a length of 2-3 μm; a characteristic feature is the presence of endospores, which are formed in the presence of oxygen, the shape is oval. Cells are located singly or in a chain.

The optimal growing conditions are as follows: temperature 37 C; The pH of the medium is 6.5-8.5.

When growing for 24-48 hours. on a solid nutrient medium (MPA), dissociation of colonies of the following variants/types is observed: B-form - flat, with a rough surface, matte, dry, with a cut edge, colony size 4-5 mm; O-shape - convex, smooth, mucous, has a flat edge, the size of the colonies is 3-4 mm; 5-form - flat, smooth, paste-like, has a slightly wavy edge, the size of the colonies is 3-5 mm.

When cultivated on liquid nutrient media (MPB, Gause medium, 10 95 wheat bran extract), bacteria form a dense surface film.

Physiological and biochemical properties. The microorganism has aerobic properties, hydrolyzes starch, reduces the viscosity of gelatin, decomposes casein, and forms 3-glucose acid. Genetic features of the strain (auxotrophicity) - non-auxotrophic. This strain produces a complex of cytotoxic lectins and proteases.

The culture fluid has antimicrobial activity up to the 5th. ashgeiv (in a dilution of 1:100), agglutinating activity to blood erythrocytes and antitumor cytotoxic and cytolytic activity relative to tumor cells of various histogenesis (adenocarcinomas

Ehrlich, Walker carcinosarcomas, Lewis lung carcinomas, B-16 melanomas).

The efficiency of bacterial biosynthesis processes is significantly influenced by environmental factors and cultivation conditions. Under different growing conditions, the same bacterial culture can completely stop the formation of certain biologically active substances, and vice versa - simultaneously produce different compounds. External factors that affect the biosynthetic activity of bacteria include the composition of the nutrient medium, the nature and concentration of carbon and nitrogen sources, the pH of the medium, as well as methods of conducting the cultivation process.

To work out the regime of cultivation of Vasiyya5 5ibBiyv IMV B-7724, the generally accepted standard medium - MPB and semi-synthetic Gause medium was used.

The age and amount of inoculum have a significant effect on the biosynthesis of cytotoxic substances. for Sowing the nutrient medium (MPB and Gauze medium) used vegetative

(20-24-hour) culture forms previously grown in stationary conditions at 37 "C.

The resulting culture was introduced into the medium for cultivation in the amount of 1.5 to 15 95 (by volume) and the intensity of the accumulation of synthesized cytotoxic substances was determined dynamically. It was established that the maximum biosynthesis of cytotoxic substances by the producer was observed when using 7-10 95 inoculum.

The optimal parameters of the biosynthesis of cytotoxic substances during the cultivation of the Vasiishv 5,yBiy5 IMV B-7724 strain in stationary conditions (thermostat) and in conditions of periodic cultivation (rocker) were practically the same. The difference was only that the growth and development (that is, the accumulation of active substances) of the producer in the thermostat was extended over a period of time.

It was established that the cytotoxic effect of the culture liquid Vasiy5 5,iryv5 IMV B-7724 on tumor cells consists of two stages: at the first stage of incubation, agglutination of tumor cells occurs, the formation of cell conglomerates; on the second - with longer incubation, lysis of tumor cells occurs. This may indicate that the culture liquid has both agglutinating and enzymatic activity, in this case proteolytic, that is, it is bifunctional, or contains at least two substances with different biological activity.

Based on this, for the cultivation of this strain, nutrient media optimized for the synthesis of lectins were studied - semi-synthetic Gause medium and a medium based on wheat bran (1095 extract), which is used for the biosynthesis of proteolytic enzymes.

During the growth of Vasiysh5 v5yIyBii5 IMV B-7724 on the studied nutrient media, the hemagglutinating activity (GAA) of the microorganism's culture fluid relative to rabbit erythrocytes was studied |4| and cytotoxic activity of IP against cells of various model tumors (ascites form of Ehrlich adenocarcinoma, sarcoma-37, Walker carcinosarcoma) (51.

Comparative study of the dynamics of the accumulation of cytotoxic substances in the culture fluid

Vasiyi5 virion IMV B-7724 showed that such substances are synthesized during the growth of this microorganism on all studied media. The peak of the cytotoxic effect on tumor cells of the culture fluid of bacteria grown on three nutrient media (MPB, Hause, 10 95 wheat bran extract) is observed on the 5-8th day of culture growth, however, the quantitative ratio of synthesized cytotoxic substances, and possibly their nature , different

Examples 1 and 2, tables (Table 1, Table 2 and Table 3) and drawings confirm the essence of a useful model.

Example 1.

The culture fluid of bacteria cultured on MPB and Gause medium in the first 10-15 minutes of incubation with tumor cells led to agglutination of 90-100 95, and at the end of incubation caused complete or partial lysis of tumor cells (Table 1, Table 2).

Table 1

Cytotoxic and agglutinating activity of the culture fluid of strains Vasiyiv5 zibiyv5 IMV B-7025 (prototype) and Vasiyiv5 5,ibriyv IMV B-7724 (applicable) when cultured on MPB medium

Doba liquid what what what declared declared declared declared tey 72 | 0 | 0 101 0 101 0 ten le | 30 | 40 | 0 | sh0 | 70 | 20 ten le | 40 | 50 | 0 | sh0 | 40 | 50 forehead YZZ«8A at 5 I 5 5 5 6 6 6 112 | 45 | 55 | 0 | 0 | 55 | 60 7.2 | 90 | 95 | 40 | 50 | 60 | 7 in shshi shi shshipshshsh shi she 712 | 90 | 95 | 90 | 9 | 85 | 9 7.12 | lo | 700 | 80 | v85 | 90 | 95 ее 712 | 70 | 80 | 40 | 50 | 90 | 95 oo atmvna YuZ0 5:35 -- 5 ---- 5 52 o 4L- 712 | 50 | 60 | 50 | 60 | 85 | 9 12 | 70 | 75 | 50 | 60 | 80 | 85 (

Table 2

Cytotoxic and agglutinating activity of the culture fluid of strains Vasiyiv5 zibiyv5 IMV B-7025 (prototype) and Vasiyiv 5,ibriy5 IMV B-7724 (applicable) when cultured on medium

Hause

Cultural - yunkNN-: what what what declares declares declares

Dt w t W TO W TO W TO W" TV NO": I 712 | 0 | 0 1 0 1 0 1 0 1 0

Dt v To C TO TO TO TO TO TT TO TO ET 712 | 0 HER 0 1 0 1 0 1 20 1 with

NN

712 | 15 | 30 | 0 | that0 1 45 1 Yuyuzh 50 712 | 20 | 50 | 20 | 50 | 60 | 7

Gag shi write shi shi shi 712 | 20 | 45 | 20 | 40 | 70 | yush 80 / 12 | 30 | 7 | 30 | 60 | 90 | 95 nn 712 | 20 | 60 | 20 | 50 | 90 | 98 / ovo aty 02082600 --0 3-00 -- 56 - 5 « -

Knish shi shi shsh shi write 12 | lo | 40 | lo | 40 | 80 | 85

To study the properties of the culture fluid of microorganisms of both strains, the hemagglutination reaction was used, which is based on the specific binding of lectins to carbohydrates located on the surface of erythrocytes, and was expressed as the titer of RHA (drawing). vzybiyiv IMV B-7025 (prototype) and Vasiyiv5 5yYibiyyiv IMV B-7724 (applicable) during cultivation on Hause medium.

The claimed strain has greater stability in the production of cytotoxic substances than the prototype strain, which is clearly visible from the graphs presented.

Example 2.

The culture fluid of bacteria grown on 10 95 wheat bran extract showed a lysing effect on tumor cells (100 95 cells) with a complete absence of agglutinating activity relative to the latter (Table 3). This may indicate the synthesis on this environment, mainly of substances with proteolytic activity of a non-lectin nature. The confirmation of this conclusion is the presence in the culture liquid of a microorganism grown on this medium, the total proteolytic activity detected electrophoretically using gelatin as a substrate |bI.

Table C

Cytotoxic and agglutinating activity of the culture fluid of strains Vasiyiv5 zibiyv5 IMV B-7025 (prototype) and Vasiyiv5 5,ibriy5 IMV B-7724 (applicable) when cultivated on 10 Fo wheat bran extract

Cultural ---- :

A day liquid ten en Shev, en declared declared declared

Dt tv no to v to V to NN pTMN NN" t V. TI 712 | 0 ІІ щ0 101 10 | 0 0

Dt niv vo stv v "to V t NN tt NN" t V. TI 712 | 10 | 20 | tho | 3 | 0 0

Dt vv nos tv V g. V t NT" I" BUT A TI 712 | 95 | sh98 | 20 | 3 | 0 0 lo obatmvna ZNA 5 5 5 5 I« 5 L5 and 712 | 98 | sh9 | 30 | 4 | 0 0

Boba FOR - 50 - 500 5 5 5 5 1 L5 and 712 | lo0 | 100 | 35 | 50 | 0 | 0

Knish shi shi shi shshsh shshsh she 712 | lo | lo | 80 | sh9 | 0 0

Dt vv no gl V p" Vl NN "t I" NO A TI 712 | lo | lo | 60 | 7 | 0 0 wool ZNA - 500-09-51 -0-- 17112 | lo | forehead | 15 | 25 | 0 | 0

Dt t tt o TT v Tov TK OVT TO 712 | 70 | 80 | 710 | 20 | 0 0 oto bative Z 565 6 5 6 o 5 0 - I 712 | 50 | 60 | 0 | 20 | 0 1 0

Thus, the claimed Vasiyshiv5 5,iriyv IMV B-7724 strain produces cytotoxic substances with antitumor properties when grown on all investigated nutrient media (MPB, Gause medium and 10 95 wheat bran extract). That is, the strain is undemanding to nutrients, which are necessary for the biosynthesis of a biologically active substance.

This property makes it possible to use available and cheap nutrient media for the cultivation of the microorganism. The culture fluid of the strain has bifunctional activity (agglutinating and cytotoxic) against tumor cells. When growing the Vasiyv5 5,yBiy5 IMV B-7724 strain on MPB and Gause media, the culture fluid agglutinated 90-100 Fo of tumor cells (which was associated with the synthesis of lectin-type substances with cytotoxic activity on these media). When growing the strain on 10 95 wheat bran extract, the culture liquid showed a 100 95 lysing effect on tumor cells (which is related to the synthesis of substances with proteolytic activity on this medium). The claimed strain has greater stability in the production of cytotoxic substances than the prototype strain.

Therefore, the claimed Vasiya5 strain of IMV B-7724 is more effective and stable in the production of cytotoxic substances, in particular, lectin and protease with high antitumor activity. The technical result of a useful model has been achieved.

Sources of information: 1. Kovalenko E.O. Extracellular lectins of bacteria of the genus VasiPsh5. Diss. ... d. biol. N. - Kyiv, 1999. 2. Patent Mo56348 Sha. The bacterial strain Vasiyi5 yYibBiiv is a producer of antitumor cytotoxic substances / Potebnya H.P., Lisovenko T.S., Cheremshenko N.L. etc. // Bull. industrial property. - 2003. - Mo 5. 3. Vegdeu5 Mapiai oi Oeiettipaiime Vasiegioiodu. Ediope 8th / Ed. Vysnapap R.E., Sirropzv K.E. - Vaitsitoge, UUShatv»v 5. MKip5, 1974. - 1268 4. Lutsik M.D., Panasyuk E.N., Lutsik A.D. Lectin. Lviv: Higher School, 1981. - 155 p. 5. J. Klaus. Lymphocyte!. Method. M.: Mir, 1990. - 395 p.

6. Moii O.M., Moipaieiv A.G., Novip5Ku K.V., Ocidieu UR. Titog rgotoieg-viittsiatey Me 92000 deiayypase 5esteiva Bu pottai apa taiidpapi pitanp seiI5. IboYayop apa sNagasieegizayop oi epgute iot NT 1080 Shtokg seiYIz // Sapseg Nez. - 1990. - M. 58, Mo. 19. - R. 6162-6170.

Fig. The dynamics of the detection of hemagglutinating lectin activity in the culture liquid of strains Vasiyv5 virii IMV B-7025 (prototype) and Vasiyv5 5!,Biy5 IMV B-7724 (applicable) during cultivation on Hause medium

Designation: along the abscissa axis - time (day), along the ordinate axis - GAA ("titer" of RGA), 77 strain - prototype, 7777 - the declared strain.

Claims (1)

FORMULA OF THE INVENTION The bacterial strain Vasiy5 z, ibiyv IMV B-7724 is a producer of cytotoxic substances with antitumor effect, which is registered in the Depository of the Institute of Microbiology and Virology named after D.K. Zabolotny National Academy of Sciences of Ukraine under number IMV B-7724. BK fretenteeentyuretestnyatya veni nyni nyni shi print mn nn y ! ! E she and I V! and N N N «7 : E ( hu E N ! EE r nn inn zi nn zenn ky a p pan ya EYA experience - nyshishshiishishshshshi she she nyshshi shishshiny nina rshishi shishi; shi SHHY shi she ; ; : i i and I E: 1; -- 1 and || iz E: chno nia for vynnykh v nn ny ny | spyateityatayaiya i V: i she: 1 i i! she | EV I i E! DK: SHY SHE I KI Melon; in pen pi pen AAltAM psh nin wines and those she we she hny shi shi na sha shSHSHYA ! Sun