Mikrobiol. Z. 2019; 81(4):3-14.
doi: https://doi.org/10.15407/microbiolj81.04.003

Extracellular Lectin Produced by Bacillus subtilis Strain IMV B-7014
Depending on the Culture Conditions

Kisten O.G.1, Kovalenko E.O.1, Getman K.I.1, Sashchuk O.V.2, Pidgorsky V.S.1, Tyshchenko L.M.3

1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

2Odesa Mechnikov National University
2 Dvoryanskaya Str., Odesa, 65082, Ukraine

3National University of Life and Environmental Sciences of Ukraine
15 Heroiv Oborony Str., Kyiv, 03041, Ukraine

The aim. To develop a new nutrient medium for the biosynthesis of extracellular lectin strain Bacillus subtilis IMB B-7014; to study changes in biomass and pH values, accumulation of protein metabolites in the culture fluid (CF) and in its foamed fraction under separate batch cultivation of the R and S morphotypes of the strain in the developed medium. Methods. Microbiological, biochemical, immunological. The selection of the source of inorganic nitrogen (ammonium citrate, ammonium nitrate, ammonium sulfate, glycine, diammonium phosphate, urea) was performed using the base medium of the following composition, g/l: galactose – 10.0; yeast extract – 2.0; K2HPO4 – 0.70; KH2PO4 – 0.30; NaCl – 0.50; MgSO4∙7H2O – 0.50; CaCl2∙6H2O – 0.10; FeSO4∙7H2O – 0.005; CoCl2∙6H2O – 0.005; MnSO4∙4H2O – 0.005; pH 7.0. Lectin activity (LA) of the CF of both morphotypes of the strain evaluated by the level of hemagglutination activity of its supernatant with rabbit erythrocytes. The molecular weight of the protein elements was determined in a denaturing system (SDS-PAGE). Results. The highest values of LA of the CF observed using ammonium sulfate. Differences in the dynamics of changes in optical density and pH of the CF during batch cultivation of the R and S morphotypes of the strain not observed, whereas the LA of the R morphotype CF was higher. Major and sub-major protein components with a molecular weight of about 50 and 73 kDa, respectively, found in the CF and in its foamed fraction of both strain morphotypes. A unique component with a molecular weight of 64.5 kDa was present in the foam. A component with a molecular weight of 84 kDa was present on the electrophoregrams of the CF R and S morphotypes in an insignificant amount, but was absent in the foam fraction. A sub-major component with a molecular weight of 43 kDa of a foam fraction found in the CF S and R morphotypes in trace amounts. The foam fraction characterized by a large presence of protein components due to their transition from the CF. Conclusions. The LA of the CF R morphotype was higher than for the S morphotype. The foam fraction contained more protein components than the CF.

Keywords: Bacillus subtilis, R and S strain morphotype, nitrogen source, extracellular lectin, culture fluid, foam, protein components.

Full text (PDF, in English)

  1. Lutsyk MD, Panasyuk EN, Lutsyk AD. [Lectins]. Lviv: Vyshcha shkola; 1981. 152 p. Russian.
  2. Podgorsky VS, Kovalenko EA, Simonenko IA. [Lectins of bacteria]. Kiev: Naukova dumka; 1992. 204 p. Russian.
  3. Sharon N, Lis H. Lectins. 2nd ed. Dordrecht: Springer; 2007. https://doi.org/10.1007/978-1-4020-6953-6
  4. Kudryavtseva IG, Sharykina NI, Kovalenko EO, Khavych OO, Pavlovska GP. [Antitumor activity and toxicity of sialic acid-specificic lectins from bacteria]. Liky. 2006; (1-2):89-93. Ukrainian.
  5. Pidgorsky VS, Kovalenko EO, Rybalko SA, Sharykina NI, Getman K.I. [Medicinal product that contains bacterial lectin for the treatment of patients with influenza]. Ukraine patent UA 83578. 2008 Jul 25. Ukrainian.
  6. Pidgorsky VS, Kovalenko EO, Rybalko SA, Sharykina NI, Getman KI. [Medicinal product that contains bacterial lectin for the treatment of patients with hepatitis C]. Ukraine patent UA 83305. 2008 June 25. Ukrainian.
  7. Pidgorsky VS, Kovalenko EO, Rybalko SA, Sharykina NI, Getman KI. [Medicinal product that contains bacterial lectin for the treatment of patients with herpes virus 1 and 2 types] Ukraine patent UA 84928. 2008 Dec 10. Ukrainian.
  8. Pidgorsky VS, Rybalko SA, Kovalenko EO, Sharykina NI, Getman KI, Maksymenok OV, et al. [Application of sialic acid-specific lectin that was isolated from Bacillus subtilis strain IMV 668 for the inhibition of reproduction of human immunodeficiency virus] Ukraine patent UA 68373. 2004 Aug 16. Ukrainian.
  9. Rudenko AO, Andreeva OG, Karlovsky OA, Rybalko SA, Dyadyun ST, Kovalenko EO, et al. [A method of treatment of herpes-viral lesions of the nervous system]. Ukraine patent UA 22155. 2007 Apr 10. Ukrainian.
  10. Pidgorsky VS, Kovalenko EO, Karpova IS, Sashchuk OV, Getman KI., Ruban TO, Sukhorada OM, Lukash LL. [The influence of lectin isoforms of Bacillus subtilis saprophytic strain IMV B-7014 on viability of normal and cancer cells in vitro]. Mikrobiol Z. 2012; 74(3):3-9. Ukrainian.
  11. Wainmright M, Canham LT, al-Wajeeh K, Reeves CL. Morphological changes (including filamentation) in Escherichia coli grown under starvation conditions on silicon wafers and other surfaces. Lett Appl Microbiol. 1999; 29:224-27. https://doi.org/10.1046/j.1365-2672.1999.00602.x
  12. Davidson CJ, Surette MG. Individuality in bacteria. Annu Rev Genet. 2008; 42:253-68. https://doi.org/10.1146/annurev.genet.42.110807.091601
  13. Wolf DM, Vazirani VV, Arkin AP. Diversity in times of adversity: probabilistic strategies in microbial survival games. J Theor Biol. 2005; 234:227-53. https://doi.org/10.1016/j.jtbi.2004.11.020
  14. Reinheimer JA, Morelli L, Bottazzi V, Suarez V. Phenotypic variability among cell of Lactobacillus helveticus ATCC 15807. Int Dairy J. 1995; 5:97-103. https://doi.org/10.1016/0958-6946(94)P1602-A
  15. Lutsyk MD, Panasyuk EN, Antonyuk VA. [Methods of searching for lectins (phytohaemagglutinins) and determination of their immunochemical specificity: Methodical recommendations for biochemists and immunologists]. Lviv; 1980. 20 p. Russian.
  16. Tareb R, Bernardeau M, Horvath P, Vernoux JP. Rough and smooth morphotypes isolated from Lactobacillus farciminis CNCM I-3699 are two closely-related variants. Int J Food Microbiol. 2015; 193:82-90. https://doi.org/10.1016/j.ijfoodmicro.2014.08.036
  17. Jakava-Viljanen M, Avall-Jaaskelainen S, Messner P, Sleytr UB, Palva A. Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions. J Bacteriol. 2002; 184:6786-95. https://doi.org/10.1128/JB.184.24.6786-6795.2002