Physical-Chemical and Cytotoxic Properties of Bacillus subtilis IMV B-7724 Extracellular Lectin

Abstract

Aim. To study the chemical composition, sugar specificity and physicochemical properties of the extracellular lectin isolated from Bacillus subtilis ІМV В-7724. Methods. Biochemical, spectrophotometric, immunological and cultural methods were used to assess the physicochemical and a number of biological properties of lectin isolated from the culture fluid of bacteria B. subtilis ІМV В-7724. Molecular weight of the lectin was estimated in polyacrylamide gel electrophoresis. Analysis of the elemental composition was done using Perkin-Elmer 2400 CHNS analyzer. Temperature and pH stability of lectin were examined based on residual hemagglutination activity of the lectin. Cytotoxic activity was determined by the MTT-assay. The statistical analysis was made using Student’s t-test. Results. B. subtilis IMV B-7724 lectin is a glycoprotein (protein – 86.0%, carbohydrates – 7.0%) with molecular weight of 18–20 kDa (major). Analysis of the elemental composition revealed that it contains 34.00% of carbon, 7.04% of hydrogen, 16.61% of nitrogen, 42.35% of oxygen. Amino acid composition analysis determined that it is rich in leucine, tyrosine and phenylalanine. The lectin exhibited high sugar-binding specificity toward N-acetylneuraminic and N-glycolylneuraminic acids (minimal inhibitory concentration – 0.3 mM for both sugars). The lectin is heat and acid stable, has long shelf life. Conclusions. These results provide the rationale to pursue further investigation for possible ways and modes of B. subtilis IMB B-7724 lectin application in clinical settings.

Features of the Synthesis of Extracellular Cytotoxic Lectin Bacillus subtilis IMV B-7724, Depending on the Cultivation Conditions in the Laboratory Fermenter

“…0.2 ± 0.1 mg/mL. In contrast to the above, a slower increase in the OD of the CF, a decrease in μ max , and signifi cant acid formation (15-fold at the 9 th h, 0.25 h −1 , and pH decrease to 5.8 units, respectively) were observed for variant 3; in this case, the level of HAA of CF was minimal (2)(3)(4) and was absent in the corresponding isolated samples . Th e probable reason for such diff erences was the limited mass transfer in the CF due to the isolating eff ect of the foam layer on its surface formed as a result of intensive agitation.…”

“…Obtaining biologically active substances of microorganisms capable of specifi c eff ects on immunogenesis, immune response, and tumor growth control remains very relevant. In particular, longterm studies of extracellular sialospecifi c lectins of saprophytic aerobic bacteria of the Bacillus genus have shown that they are characterized by a selective eff ect on tumors of various origins, are inducers of natural gamma-interferon synthesis [1][2][3][4], as well as active inhibitors of adsorption and reproduction of infl uenza, herpes, hepatitis C, and HIV viruses [5][6][7][8]. Th e presence of such unique properties opens up broad prospects for the use of these substances in biology and medicine, in particular for the development of new biotherapeutic agents to improve the eff ectiveness of anticancer therapy.…”

“…Accordingly, the highest HAA (128 hemagglutinating units (HAU)) was observed in CF samples obtained within 2-5 days, whereas the maximum yield of the preparation (80 mg/L) with HAA of 2048 HAU and cytotoxic activity (IC -95.0 ± 2.0%) was obtained on the 4 th day of strain growth. Further study of the chemical composition, carbohydrate specifi city, and physicochemical properties of the extracellular cytotoxic lectin (ECL) of strain IMV B-7724 revealed that the obtained bacterial lectin was a glycoprotein with a molecular mass of 18-20 kDa with the highest affi nity to Nacetylneuraminic and N-glycolylneuraminic acids, high thermal stability, resistance to changes in pH, and long-term storage [4]. In this work, CF for subsequent lectin isolation was obtained by growing the strain in shaking fl asks, which is usually used in laboratory studies, but the results obtained in this way cannot be directly extrapolated to the possible productivity of a similar process on a larger scale, usually performed in fermenters (bioreactors) with a stirrer.…”

Оцінка гострої токсичності лектину B. subtilis ІМВ B-7724

“…Досліджуваний нами лектин було виділено з культуральної рідини мікроорганізму B. subtilis IMB B-7724; у дослідженнях in vitro охарактеризовані його фізико-хімічні та біологічні властивості. Показана його цитотоксична активність відносно пухлинних клітин, що вказує на перспективність дослідження можливості його використання як засобу протипухлинної терапії [11]. На перших етапах дослідження нових біологічно активних речовин необхідне проведення оцінки параметрів гострої токсичності за умови одноразового введення речовини [12].…”

“…За парентерального (п/ш і в/о) введення лектину використані наступні дози: 125, 250, 375, 500 мг/кг ваги тварини; перорально лектин вводили тільки в найбільшій дозі-500 мг/кг. Підбір використаних доз базувався на результатах попередніх досліджень біологічних властивостей лектину in vitro [11] та на пiдcтавi даних лiтератури щодо цитотоксичних властивостей та активності позаклiтинних лектинів сапрофiтних бактерiй роду Bacillus [10]. Досліджувану речовину вводили одноразово, в об'ємі 1 мл на мишу (в/о і п/ш), 0,8 мл на мишу (per os).…”