Izolacija in karakterizacija zunajceličnega polisaharida EpsA-O pri bakteriji Bacillus subtilis

Krajnc, Mojca (author), Dogša, Iztok (mentor)

Pri bakteriji Bacillus subtilis je tvorba biofilmov eden izmed najpomembnejših prilagoditvenih procesov. Tvorbo biofilmov omogočajo zunajcelične polimerne snovi (EPS), katerega sestaven del je polisaharid EpsA-O, ki je nujen za tvorbo zrelega biofilma. Z gojenjem visokoproduktivne mutante seva B. subtilis (?sinR) na trdnem gojišču MSgg s povečano koncentracijo glicerola smo po 24 urni inkubaciji pridobili zunajcelični material iz katerega smo izolirali polisaharid EpsA-O. Kot negativna kontrola pridobitve polisaharida EpsA-O nam je služila mutanta seva B. subtilis ?epsA-O. Osredotočili smo se na čistost, reologijo in HPSEC analizo izolatov. Na mikroskopskem, makroskopskem in biokemijskem nivoju smo opazili razlike med visokoproduktivnim sevom in negativno kontrolo. Razlike v izoliranem materialu sevov ?sinR in ?epsA-O so prisotne tudi na kromatogramih, ki smo jih dobili z metodo HPSEC. Kromatogram refrakcijskega indeksa seva ?sinR ima dodaten vrh, za katerega sklepamo, da pripada polisaharidu EpsA-O in spada v velikostni razred > 3000 kDa. S spremljanjem čistosti izolatov smo ugotovili, da največ nečistot predstavljajo biološke molekule, ki absorbirajo svetlobo v UV delu spektra, ključen korak čiščenja predstavlja sprememba pH raztopine. Z reološkimi metodami smo potrdili, da je viskoznost raztopin izolatov večja, če smo bakterije gojili na trdnem in ne na tekočem gojišču. Prav tako smo ugotovili, da je obnašanje raztopin izolatov seva ?sinR psevdoplastično do zelo nizkih koncentracij, medtem ko se izolati iz seva ?epsA-O obnašajo kot Newtonske tekočine. Visoka intrinzična viskoznost je v skladu s HPSEC velikostno oceno polisaharida EpsA-O. Z oscilacijskim testom smo ugotovili, da prečiščen izolat pri koncentraciji 1 % pridobi strukturne lastnosti gela, vsi vzorci pod to koncentracijo imajo strukturne lastnosti viskoelastičnih tekočin.

• Language: Slovenian
• Keywords: Bacillus subtilis, EPS, polisaharid EpsA-O, biofilm, čistost, reologija
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [M. Krajnc]
• Year: 2019
• PID: 20.500.12556/RUL-110440
• UDC: 602.3:579.852.11:604.2:547.458(043.2)
• COBISS.SI-ID: 9363065
• Publication date in RUL: 14.09.2019

KRAJNC, Mojca, 2019, Izolacija in karakterizacija zunajceličnega polisaharida EpsA-O pri bakteriji Bacillus subtilis [online]. Master’s thesis. M. Krajnc. [Accessed 15 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=110440

Secondary language

• Language: English
• Title: Isolation and characterization of extracellular polysaccharide EpsA-O from Bacillus subtilis

Abstract

Biofilm formation is one of the most important adaptation processes in Bacillus subtilis. The exopolymeric substances (EPS) are crucial element in the process of biofilm formation. The essential EPS component is polysaccharide EpsA-O, which is necessary for the formation of mature biofilm. With the cultivation of highly productive mutant strain B. Subtilis (ΔsinR) on the solid MSgg medium for 24h with increased glycerol concentration, we obtained extracellular material from which the polysaccharide EpsA-O was isolated. For negative control we used mutant strain B. subtilis ΔepsA-O. We have focused on purity, rheology and HPSEC analysis of isolated material. At the microscopic, macroscopic and biochemical levels, we observed differences between the highly productive strain and the negative control. The differences between the isolated materials of strains ΔsinR and ΔepsA-O are also evident on the chromatograms obtained by HPSEC method. The refractive index chromatogram from the strain ΔsinR contains an additional peak, which is assumed to belong to an EpsA-O polysaccharide. We estimated that it belongs to the size class > 3000 kDa. By monitoring the purity of isolates, we found that the most impurities represent biological molecules that absorb light in the UV part of the spectrum, the most important step of purification process represents a change in the pH of the solution. With the rheological methods, we confirmed that the viscosity of isolates solutions was much higher if the cells were cultivated on a solid and not on the liquid medium. We also found that the behaviour of isolates solutions from strain ΔsinR is pseudoplastic even at very low concentrations, while isolates from the strain ΔepsA-O behave like Newtonian fluids. High intrinsic viscosity supports the estimated HPSEC of the EpsA-O polysaccharide. By oscillatory assay we found that the purified isolate at a concentration of 1% has a gel-like structure, all samples below this concentration have liquid-like structure.

• Keywords: Bacillus subtilis, EPS, polysaccharide EpsA-O, biofilm, purity, rheology