ISSN 2410-7751 (Print)
ISSN 2410-776X (Online)
"Biotechnologia Acta" V. 8, No 5, 2015
https://doi.org/10.15407/biotech8.05.048
Р. 48-54, Bibliography 15, English
Universal Decimal Classification: 541.49:546.47
BACILLUS THURINGIENSIS ELASTASES WITH INSECTICIDE ACTIVITY
1 Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, Kyiv
2 Opole University, Faculty of Natural Sciences and Technology, Opole, Poland
The purpose of the research was a screening of proteases with elastase activity among Bacillus thuringiensis strains, their isolation, partially purification, study of physicochemical properties and insecticide activity in relation to the larvae of the Colorado beetle. The objects of the investigation were 18 strains of B. thuringiensis, isolated from different sources: sea water, dry biological product "Bitoksibatsillin" and also from natural populations of Colorado beetles of the Crimea, Kherson, Odesa, Mykolaiv and Zaporizhiia regions of Ukraine. Purification of enzymes with elastase activity isolated from above mentioned strains was performed by gel-chromatography and insecticide activity was studied on the 3–4 larvae instar of Colorado beetle. The ability of a number of B. thuringiensis strains to synthesize the proteases with elastase activity has been established. The most active were enzymes obtained from strains IMV B-7465, IMV B-7324 isolated from sea water, and strains 9, 902, Bt-H and 0-239 isolated from Colorado beetles. The study of the physicochemical properties of the partially purified proteases of these strains showed that they belonged to enzymes of the serine type. Peptidases of a number of B. thuringiensis strains (IMV B-7324, IMV B-7465, 902, 0-239, 9) are metal-dependent enzymes. Optimal conditions of action of all tested enzymes are the neutral and alkaline рН values and the temperatures of 30–40 °С. The studies of influence of the complex enzyme preparations and partially purified ones of B. thuringiensis strains on the larvae instar of Colorado beetles indicated that enzymes with elastase activity could be responsible for insecticide action of the tested strains.
Key words: Bacillus thuringiensis, elastase activity, insecticide activity.
© Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, 2015
References
1. Kandibin N. V., Patyka T. I., Ermolova V. P., Patyka V. F. Micro biocontrol of population of insects and dominant Bacillus thuringiensis. Ed. Kandibin N.V. PublisherVIZR, St. Petersburg, Pushkin. 2009. 252 p. (In Russian).
2. Patyka V. F., Patyka T. I. Ecology of Bacillus thuringiensis. PGAA, Kyiv. 2007, P. 120–128. (In Ukrainian).
3. Palma L., Munoz D., Berry C., Murillo J., Caballero P. Bacillus thuringiensis toxins: an overview of their biocidal activity. Toxins. 2014, V. 6, P. 3296–3325.
http://dx.doi.org/10.3390/toxins6123296
4. Liu X. Y., Ruan L. F., Hu Z. F., Peng D. H., Sao of S. Y., Yu Z. N., Liu Y., Zheng J. S., Sun M. Genome-wide screening reveals the genetic determinants of antibiotic insecticide in Bacillus thuringiensis. J. Biol. Chem. 2010, V. 285, P. 39191–39200.
http://dx.doi.org/10.1074/jbc.M110.148387
5. Barboza-Corona J. E., Delgadillo-Angeles J. L., Castaneda-Ramirez J. C., Barboza-Perez U. E., Casados-Vazquez L. E., Bideshi D. K., del Rincon-Castro M. C. Bacillus thuringiensis subsp. kurstaki HD1 as and factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use. Microb. Cell Fact. 2014, 13 (15). doi: 10.1186/1475-2859-13-15.
6. Luo X., Chen L., Huang Q., Zheng J., Zhou W., Peng D., Ruan L., Sun M. Bacillus thuringiensis metalloproteinase Bmp1 functions as and nematicidal virulence factor. Appl. Environ. Microbiol. 2013, 79 (2), 460–468.
http://dx.doi.org/10.1128/AEM.02551-12
7. Harrison R. L., Bonning B. C. Proteases as insecticidal agents. Toxins. 2010, V. 2, P. 935–953.
http://dx.doi.org/10.3390/toxins2050935
8. Маtseliukh O. V., Nidialkova N. A., Varbanets L. D. Purification and physico-chemical properties of Bacillus thuringiensis IMV B-7324 peptidase with elastolytic and fibrinolytic activity. Ukr. Biochem. J. 2012, 84 (6), 25–36. (In Ukrainian).
9. Маtseliukh О. V., Varbanets L. D., Ivanytsia V. О. 2012. Strain Bacillus thuringiensis IMV В-7324 – producer of extracellular elastase. UA Patent 97906 C2, March 26 2012. (In Ukrainian).
10. Маtseliukh O. V. Substrate specificity of Bacillus thuringiensis IMV B-7324 serine alkaline peptidase. Microbiology and Biotechnology. 2014, 2 (26), 24–33. (In Ukrainian).
11. Маtseliukh O. V. Obtaining of mutants of Bacillus sp. with enhanced elastase production. Biotechnology. 2010, 3 (2), 42–47. (In Ukrainian).
12. Koltukova N. V., Vaskivniuk V. T. Selection of the methods for isolating the proteolytic enzymatic complex from Bacillus mesentericus 316m in stab culture. Mikrobiol. Zn. 1980, 42 (2), 245–249. (In Russian).
13. Trombridg G. O., Moon H. D. Purification of human elastase. Proc. Soc. Exp. Biol. Med. 1972, 141 (13), 928–931.
http://dx.doi.org/10.3181/00379727-141-36903
14. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193 (1):265–275.
15. Brar S. K., Verma M., Tyagi R. D., Surampalli R. Y., Barnabe S., Valero J. R. Bacillus thuringiensis proteases: Production and role in growth, sporulation and synergism. Proc. Biochem. 2007, 42 (5), 773–790.
http://dx.doi.org/10.1016/j.procbio.2007.01.015
16. Nіdialkova N. A., Varbanets L. D., Іvanitsa V. O. A bacterial strain Bacillus thuringiensis var. israelensis – producer of extracellular collagenase. UA Patent 97906. January 26 2015. (In Ukrainian).
17. Salazar O., Asenjo J. A. Enzymatic lysis of microbial cells. Biotechnol. Lett. 2007, 29 (7), 985–994.
http://dx.doi.org/10.1007/s10529-007-9345-2