Study of enzymatic activity of metabolic complex of Bacillus subtilis for development of enzyme preparations

Results of screening the general hydrolytic, amilolytic, proteolytic activities of strains Bacillus subtilis isolated from soil. The effect of pH, temperature, type of cultivation, substrate on the enzymes activities of isolated strains were studied.

Рубрика Биология и естествознание
Вид статья
Язык английский
Дата добавления 23.05.2018
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Study of enzymatic activity of metabolic complex of Bacillus subtilis for development of enzyme preparations

bacillus strain cultivation

Madybekova G.M.

The paper contains data of results of screening the general hydrolytic, amilolytic, proteolytic activities of strains Bacillus subtilis isolated from soil. The effect of pH, temperature, type of cultivation, substrate on the enzymes activities of isolated strains were studied. It was established that optimal pH was 9, temperature - 500C, the optimal growth were achieved at deep cultivation, and at use of beer pellet as a substrate.

Keywords. Bacillus subtilis, microorganisms strains, biologically-active substances, enzymes, metabolites, beer pelle, substrate.

Recently,biotechnologyare increasingly usedproducersof biologically active substances, enzymes, antibiotics,hormones, etc. Currently, the mostperspectiveand widely usedinsecticidesarebiological productsbased onbacteriaBacillus subtilisand itsmetabolites.

At the present stage of medical biotechnology, new data justifying the use of saprophytic microflora, which is capable during of their life release biologically active substances (BAS), enzymes, antibiotics, hormones, etc., inhibiting the growth of pathogenic micro-organisms, cancer and normalizing various pathological and biochemical processes in the human body, and also used as plant protection products. [1] Currently, the most perspective and widely used are biological products based on bacteria Bacillus subtilis and its metabolites.

Species Bacillus attract the attention of researchers for a long time. The accumulated knowledge in the field of microbiology, physiology,

biochemistry, and genetics of bacteria testifies about benefits of Bacillus as a producers of biologically active substances: enzymes, antibiotics, insecticides [2-5]. High adaptability to various conditions of existence (presence or absence of oxygen, the growth and development in a considerable range of temperatures, the use of various organic or inorganic compounds, etc. as sources of nourishment) contribute to the spread of germs in the soil, water, air, foodstuffs and other environmental objects, as well as in humans and animals.

A variety of metabolic processes, genetic and biochemical variability, resistance to lytic and digestive enzymes, have served as justification for the use of bacilli in the various fields of medicine [6-10].

Analysis of the results of research carried out in our country and abroad, testifies about scales of use of the Bacillus bacteria to produce products from biomass of bacteria or their metabolites. Known methods for culturing bacteria of the species Bacillus are the basis for the technology of obtainment of a number of bacterial and enzyme preparations [11-15].

On the basis of live bacteria of the species Bacillus, had been created biological products that are harmless for the microorganism, and can be used to protect plants from disease.

The aim of this work is selection of microorganism Bacillus subtilis strain from soil of South Kazakhstan region and study the complex metabolite, the metabolite activity of isolated strain, as well as the hydrolytic activity and biological properties of isolated strains of B.subtilis and to evaluate the possibility of their use for the development of the original biological product.

Creation of optimal conditions for the growth of bacteria Bacillus subtilis have essential meaning for the creation of biological products on their basis.

The medium was prepared by weighing the following medium composition in grams per litre; Bacteriological peptone-6g, MgSO4.7H2O-0.5g, KCL-0.5g, substrate-1.0g. The above medium composition were dissolved in 1000ml of distilled water after which 100ml of the medium was measured into a conical flask (250ml capacity each) heated on hot plate to homogenize and then sterilized in an autoclave at 121? for 15 minutes after which they were removed and allowed to cool before the organism was inoculated.

The screening of amilolytic activity of B. Subtilis were used Louria-Bertoni medium with 1% of starch solution.

Hydrolytical activity is determined in supernatant from cultures, which are obtained by centrifugation of all volume of culture. Asapolysaccharide substrates are used potato starch, also extract of beer pellet.

For estimation of proteolytic activity were used hydrolysis of 1% casein solution by proteases.

Figure 1shows the effect of pH on the activity of enzymes produced by Bacillus subtilis. The optimum pH was 9 for amilolytic and proteolytic activities with a concentration of 1,15 and 0.85 mg/ml/sec. accordingly.

Figure 1.Effect of pH on the activity of enzymes produced by B. subtilis.

Protease; 2 - Amilase

Figure 2 shows the effect of temperature on the activity of enzyme produced by B. subtilis. The optimum temperature for the activity of B. subtilis was recorded at 50? with a concentration of 1.15 and 0,85 mg/ml/sec.

Figure 2.Effect of temperature on the activity of enzymes produced by B. subtilis.

1 - Protease; 2 - Amilase

Isolated strains Bacillus subtilis were studied on ability to hydrolizate the plant polymeric carbohydrates, as a source was used the beer pellet.

Figure 3 showed results of supernatant hydrolytic activity determination. As a substrate for determination of summary hydrolytic activity was used beer pellet. It was established that at deep cultivation the value of hydrolytic activity of strain is similar. Maximum of activity were observed on 2nd day of cultivation; Coimparison of hydrolytic activity of film and deep cultures shows that more efficiency is deep cultivation.

Figure 3. General hydrolytical activity of strains at deep (1) and film (2) cultivation with use of extract of beer pellet

More slowly growth of hydrolases enzymes activity is explained by additional time necessary for formation of film.

Besides summary hydrolytic activity with use of beer pellet as a substrate for comparison was determined the hydrolytic activity of strains toward potato starch (table 1).

Table 1 - Activity of hydrolases in the deep cultures of strain

Strain

Cultivation time

Rate of formation of reaction products (mkmol/min. at hydrolysis of various substrates)

Starch

Extract of beer pellet

Isolated strain

72 h., deep

15,6

26,1

So, researches of general hydrolytic, amilolytic and proteolytic activity of film and deep cultures of strain testifies about possibility of deep cultivation for production of enzymes of hydrolytic complex at use the beer pellet as a substrate.

References

1. ПодберезныйВ.В., Полянцев Н.И., Ропаева Л.В.Культивирование производственных штаммов Bacillussubtilis в подсырной сыворотке // Ветеринария.- 1996. -№ 1-С. 21-29.

2. БлинковаЛ.П., Семенов С.А., Бутова Л.Г. и др. Антагонистическая активностьсвежевыделенныхштаммов бактерий рода Bacillus // ЖМЭИ. 1994. -№5. -С. 71-72.

3. ПоспеловаВ.В., Рахимова Н.Г., Халенева М.П. и др. Новые сферы применения микробных биопрепаратов для коррекции бактериоценоза организма человека.//Иммунобиол. препараты. М. -1989. -С. 142-152.

4. СорокуловаИ.Б. Сравнительное изучение биологических свойств био-спорина и других коммерческих препаратов на основе бацилл // Мшробю-лопчний журнал. -1997. - Т. 69, -№6. - С. 43-49.

5.Buchell M.E., Smith J., Lynch H.C. A phisiological model for the control of erytromycin production in batch and cyclyc fed batch cuiture // Microbiology. -1997. V. 143, -№ 2. - P. 475-480.

6. БойкоН.В., ТуряницаА.И., ПоповичЕ.П.,ВьюницкаяВ.А. Антагонистическоедействиекультур Bacillus subtilis набактерииродаKlebsiella / Микробиол 1989. -Т. 51, -№ 1.-С. 87-91.

7. КудрявцевВ.А.,Сафронова JI.A., Осадчая А.И. и др. Влияние живых культур Bacillussubtilis на неспецифическую резистентность организма // Мик-робиол. - 1996 - Т.58, -№ 2. - С. 46-53.

8. МитрохинС.Д., ШендеровБ.А.Микробиологические и биохимические показатели изменения микробной экологии толстой кишкикрыспод влиянием рифампицина. Антибиотикиихимиотерапия- 1999, Т. 34 №6 (482-4).

9.Cromwick A.M., Birrer G.A., Gross R.A. Effects of pH and aeration on y-poly (glutamic acid) formation by bacillus licheniformis in controlled batch fermentor cultures // Biotechnol. andBioeng. 1996. - V. 50, № 2. - P. 222-227.

10. Lin S.-C., Carswell K.S., Sharma M.M., Georgiou G. Continuos production of the lipopeptidebiosurfactant of Bacillus licheniformis JF-2 // Appl. Microbiol, andBiotechnol. 1994. -V. 41, №3. -P. 281-285.

11.ЗинкинВ.Ю. Фотометрический НСТ-тест с нейтрофилами крови человека и его клинико-иммунологическая значимость у больных с травмой опорно-двигательного аппарата. Автореф. дис. канд. мед.наук. - Москва, 2004.

12 DonovanW.P., RuparM.J., SlaneiA.C. Bacillusthuringiensiscrytic, proteintoxictocoleopteranisects // Патент N 5378625СШАA61K 31/00. Опубл. 03.01.95.

13 Kubo Kazuhiro. Pure culture of Bacillus subtilis FERM BP-3418 // Пат. N 5364738. США.МКИA01N 25//00. - публ. 15.11.94.

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