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Optimization of the Production of Lipases by Bacillus subtilis
Slavíčková, Radka ; Omelková, Jiřina (referee) ; Hermanová, Soňa (advisor)
In this thesis, optimization of production of lipolytic enzymes by submerzed cultivation of Bacillus subtilis (BS) was studied. Production of lipolytic enzymes was tested in three nutrient media, which differed mainly in main sources of carbon, respectively of nitrogen. The first medium contained mainly extract from calf brain and beef heart (BHIB), the second medium contained peptone and yeast extract (NB) and the third one contained peptone and yeast extract with the addition of 2% (w/v) glucose (NBG). The highest lipolytic activity (0.0784 Uml-1) was measured in NBG medium. Maximum of lipolytic activity was observed before the end of the exponential phase of BS growth in all the media. Temperature optimum in NBG medium was determined from 30 to 50 °C, pH optimum in the range of 5 to 11 and subsequently the temperature stability of lipolytic enzymes produced by the BS was estimated. The activity value was determined spectrometrically using p-nitrophenyllaurate as a substrate. Produced lipolytic enzymes showed maximum activity at 37 °C in the alkaline pH of 8.0. Measurement of temperature stability showed that lipolytic enzymes are relatively thermostable enzymes retaining 100 % of the activity even after 1 hour of cultivation at 30 - 50 °C. The presence of 1% (w/v) olive oil in medium NBG caused a decrease in lipolytic activity by 65 % as well as in pH from 6.5 to 5.4 after 14 days of cultivation. After substitution of glucose by fructose in medium NBG, lipolytic activity showed comparable values during the first week of cultivation. On the other hand, the decrease of lipolytic activity by 29 % in the medium with fructose was observed after 14 days of cultivation. A procedure for the identification of lipolytic enzymes of BS by peptide massfingerprinting was developed to understand the potential of synthetic polyester - poly(e-caprolactone) as a lipase inductor. Degradation study of commercial polyester poly(e-caprolactone) was carried out by submerged cultivation of Bacillus subtilis in NBG medium at initial pH 7.0 and 30 °C for 14 days. PCL (Mn = 10,000, Mw = 14 000) was studied in the form of films (1.0 x 1.0 cm), which were prepared by melt-pressing, rapid cooling of the melt to 4 °C and evaporation of the solvent from 2 % dichlormethane solution. The evaluation of the films shown occurrence of weight loss (7.8 - 17.0 wt.%) together with the formation of numerous holes and cracks in the sample surface in relation to the method of the films preparation. Lipolytic activity values increased by 9 - 17 % in the degradation media compared to control samples. Densitometric monitoring showed also higher increase in cell mass in the degradation medium compared with control samples. Based on the results obtained, the degradation process induced by BS could be suggested.
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Microbial Degradation of Polycaprolactone-based Materials
Damborský, Pavel ; Stratilová, Eva (referee) ; Hermanová, Soňa (advisor)
Diplomová práce se zabývá vlivem nutričních a aeračních faktorů na produkci lipáz bakterií Bacillus subtilis (CCM 1999). Produkce lipáz byla studována zejména z hlediska katalytického působení lipáz při degradaci polyesterových řetězců. Mezi studované parametry patřily: růst bakterií, lipolytická aktivita, pH optimum, teplotní optimum, tepelná stabilita, proteolytická aktivita, množství bílkovin, atd. a to v různých typech živných medií zaočkovaných Bacillus subtilis. Jedna série vzorků kultivačních médií pro BS na bázi: pepton a kvasničný extrakt (NB), pepton, kvasničný extrakt s 2% přídavkem (w/v) glukózy (NBG) a minerální médium s kvasničným extraktem (MS-YE) obsahovala jeden PCL vzorek o definovaných rozměrech (Mn = 10 kDa, = 1.4). Experimenty probíhaly po dobu 21 dnů pří rychlosti třepání 160 a 200 rpm. Přítomnost PCL způsobila v obou typech médií (NB, NBG) inokulovaných BS zvýšení lipolytické aktivity, což naznačuje, uvolnění a následné uplatnění se nízko-molekulekulárních řetězců PCL jako substrátů pro BS. BS kultivovaný v MS-YE medium vykazoval ve srovnání s NB a NBG médii nízké hodnoty lipolytické aktivity a to i v přítomnost PCL. Během experimentů se hodnota pH posunula z neutrální (pH 7.0) do alkalické (pH 8.5-9.3) oblasti a to ve všech typech médií s i bez přítomnosti PCL vzorku v důsledku metabolických pochodů BS využívajících různé substráty. Lipolytické enzymy stanovené v supernatanech bez bakteriálních buněk vykazují dvě pH optima v přítomnosti PCL, pH 7 a 9. V nepřítomnosti polymeru vykazují pouze jedno pH optimum při pH 7. Na základě měření tepelné stability bylo prokázáno, že extracelulární lipázy jsou relativně termostabilní enzymy, zejména v nepřítomnosti polymeru. Dále byla provedena základní proteomická analýza lipáz produkovaných bakterií Bacillus subtilis v NBG médiu pomocí metody peptidového mapování (PMF). Byla ověřena přítomnost proteinů s molární hmotnosti (19.3 kDa) pomocí FPLC. SDS-PAGE a IEF-PAGE prokázaly přítomnost těchto proteinů v obou studovaných mediích inokulovaných BS (NBG vs. NBG/PCL). Zásadní rozdíly proteinového složení v přítomnosti PCL nebyly potvrzeny a identifikace pomoci MALDI-TOF hmotnostní spektrometrie nestanovila žádnou lipázu. Proces degradace v PCL vzorcích byl vyhodnocen také na základě hmotnostních úbytků, které byly zjištěny ve všech typech médií inokulovaných BS pravděpodobně v důsledku synergického účinku enzymaticky-katalyzované a biotické hydrolýzy v alkalickém prostředí. . Modelová degradační studie PCL a jeho kompozitu s oxidem grafenu (2.7 hm.%, GO) byla provedena v přítomnosti bakterie Bacillus subtilis v NBG při 30 °C a počátečním pH 7 po dobu tří týdnů. Hmotností úbytky PCL filmů se postupně zvyšovaly během celého degradačního testu až ke 12 hm%. Degradace PLC/GO kompozitu probíhala pomaleji, což je prokázáno maximální hmotnostním úbytkem 5 hm%. Podobný charakter elučních křivek PCL a jeho kompozitu stanovený pomocí SEC potvrzoval snížení molární hmotnosti po degradaci.
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Characterization of organic nanoparticles with encapsulated antimicrobial peptides
Vejrostová, Petra ; Němcová, Andrea (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on characterization of particles containing encapsulated antimicrobial enzyme lysozyme. The theoretical part deals with characterization of antimicrobial peptides and their description. Further part of review was focused on lysozyme, the selected representative used in this thesis, its structure, mechanism of action and possible usage. In the experimental part the Hartree-Lowry method was used for lysozyme detection, determination of encapsulation efficiency and for detecting the amount of lysozyme released after incubation in model physiological environment and in model foods. In process of encapsulation the highest amount of lysozyme was packed into 1% chitosan particles, manually prepared alginate particles and into liposomes. During study of stability of particles in model foods as the least stable manually prepared chitosan particles were found. The released lysozyme exhibited changes probably caused by its degradation. The highest influence on particles proved 3% acetic acid. During studying the stability of the particles in artificial digestive fluids as the most unstable manually prepared chitosan particles were found, while alginate particles were the most stable. The thesis also deals with changes in antimicrobial activity of encapsulated lysozyme in prepared particles and after its application into the model environments. A gram-positive bacteria Bacillus subtilis was used in order to test the antimicrobial activity. Antimicrobial tests showed that after encapsulation antimicrobial activity of lysozyme was substantially decreased in most samples. Size and stability of prepared particles was tested using dynamic light scattering.
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Study of Sterilization Effects Initiated by Dielectric Barriere Discharge
Slámová, Jitka ; Pekárek, Stanislav (referee) ; doc. RNDr. Karol Hensel, Ph.D., oponent (referee) ; Krčma, František (advisor)
The overall goal of the presented dissertation thesis was to study the sterilization efficiency of dielectric barrier discharge operated at atmospheric pressure. The fungi Aspergillus niger, gram-positive bacteria Bacillus subtilis and in some experiments also gram-negative bacteria Escherichia coli were used as a bio-indicator enabling to evaluate the effect of plasma assisted microbial inactivation. The samples of microorganism were placed on paper Whatman 1 or PET foil and exposed to plasma. The plasma was generated in argon, nitrogen, synthetic dry/humid air with frequency up to 10 kHz and plasma power density in the range of 1,2-2,9 W/cm3 (according to the process gas). The influence of process gas, plasma power density, plasma exposition time, type of microorganism and material of the substrate on the sterilization effect of dielectric barrier discharge was evaluated. Furthermore the contribution of each single mechanism (UV radiation, temperature and reactive species) to the sterilization effect of plasma and influence of gas humidity was evaluated. The DBD was analysed by means of optical emission spectroscopy, thermocouple was used to measure temperature during a sterilization process. In order to verify the mechanical damage of the microbial cell or the substrates during the plasma process the samples were studied by scanning electron microscopy. Generally, on the basis of experimental results, at increasing treatment times, the remaining number of spores (CFU) decreased. Similarly at increasing the plasma power input, the sterilization rate increased. When sterilising the spores of A. niger in plasma using different process gasses, the efficiency of plasma sterilization decreased as follows: argon, humid synthetic air, nitrogen and dry synthetic air. The results observed in argon plasma using different microorganism demonstrated that the sensitivity of vegetative cells resp. spores to DBD decreased as follows: A. niger spores, B. subtilis vegetative cells, E. coli vegetative cells and B. subtilis spores. Simultaneously results observed for sterilization of spores and vegetative cells of B. subtilis and A. niger demonstrated that the spores are generally more resistant to plasma than are the corresponding vegetative cells. Combining the results of contribution of each single mechanism, optical emission spectroscopy and inactivation characteristic it was found out that the reactive species significantly contribute to the plasma sterilization in all process gasses. Furthermore the inactivation process can be partly assisted by UV radiation and also the temperature can contribute in limited extent to inactivation process in some gasses. The contribution of UV radiation to the plasma sterilization decreased as follows: nitrogen, argon, dry syntetic air and humid syntetic air. Moreover it was found out that the contribution of each single mechanism can be species dependent, this is due to the different response of microorganism to the unfavorable external conditions. SEM analysis of the substrates prooved the etching actions of the plasma generated in all process gasses on the surface of the PET foil. The several minute plasma exposition of the PET foil resulted in the occurence of the „hole corrosion“ on the PET surface. Contrary to these there were no visible changes observed in the paper structure.
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Antimicrobial activity of carbon-based fillers
Stuchlíková, Olga ; Hermanová, Soňa (referee) ; Voběrková, Stanislava (advisor)
Diplomová práce se zabývá vlivem uhlíkatého plniva na životaschopnost a produkci extracelulárních látek vybrané bakterie Bacillus subtilis (CCM 1999) a kvasinky Yarrowia lipolytica (CCY 29-26-52). Antimikrobiální aktivita těchto částic, přítomných v kultivačním mediu, byla sledována pomocí následujících parametrů: růst daného mikroorganismu, produkce extracelulárních proteinů a v poslední řadě byla monitorována produkce extracelulárních polymerních substancí, které mají úzkou souvislost s tvorbou biofilmu. Suspenze materiálů (0,135 mg/mL) byly připraveny ve dvou rozdílných kultivačních mediích; tzn. živné medium s obsahem glukózy pro Bacillus subtilis a bazální medium s přídavkem Tweenu 80 pro Yarrowia lipolytica, a media byla inokulována příslušným typem mikroorganismu. Experimenty probíhaly po dobu 6 dnů při rychlosti třepání 160 rpm a teplotě 30 °C pro Bacillus subtilis a 28 °C pro Yarrowia lipolytica. Testovány byly celkem tři typy uhlíkatého nanomateriálu, získané z Katedry anorganické chemie, Vysoké školy chemicko-technologické v Praze. Tyto materiály specifikované jako materiál “A”, “B” a “C” se navzájem lišily velikostí částic a stupněm oxidace. Na základě skríningových studií byla vybrána koncentrace testovaného materiálu 0,135 mg/mL a rychlost třepání 160 rpm. Metodou měření optické hustoty vzorku při 600 nm byly sestaveny a porovnány růstové křivky obou mikroorganismů v přítomnosti testovaných nanočástic po dobu 5 dní. Tímto způsobem bylo zjištěno, že přítomnost nanočástic v mediu nemá velký vliv na růst zkoumaného mikroorganismu. Tato metoda, je však pouze orientační, protože se nevyhneme chybě díky přítomnosti mrtvých buněk. Dále byla testována produkce celkových a extracelulárních proteinů daným mikroorganismem v přítomnosti testovaných nanočástic. Nebyla však pozorována výrazná odchylka hodnot od hodnot kontrolního vzorku, který neobsahoval testovaný materiál. Na základě metod počítání kolonií (Bacillus subtilis) a buněk (Yarrowia lipolytica) byly určeny ztráty životaschopnosti mikroorganismu ve 3 časech (6, 48 a 144 hodin); v kratším časovém intervalu byl růst spíše podporován. Dále byla monitorována produkce extracelulárních polymerních substancí (EPS), tedy proteinů, redukujících substancí a polysacharidů. Tyto látky byly vylučovány daným mikroorganismem do prostředí v průběhu 24 hodin. Bacillus subtilis produkoval EPS ve větší míře než Yarrowia lipolytica. Předpokládáme, že produkce EPS by mohla souviset s tvorbou biofilmu, který chrání buňky před toxicitou nanočástic.
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Antimicrobial effect of plant extracts
Konderla, Patrik ; Vespalcová, Milena (referee) ; Veselá, Mária (advisor)
The purpose of this bachelor thesis was to research the possible antimicrobial activity of various extracts of rosehip tea and also determine the antioxidant activity and the concentration of biologically active compounds (polyphenols and flavonoids) in these tea. Teoretical part describes basic information about plant Rosa canina and chemical composition of rosehips, summary of the natural biologically active compounds and general classification of tea. The practical part is focused on detection of antimicrobial activity of tea extracts against bacterial strains Serratia marcescens and Bacillus subtilis as well as spectrophotometrical determination of concentration polyphenols, flavonoids and overall antioxidant activity of these extracts. From the results flow the testing aqueous extracts rosehips tea analysed antimicrobial effects. Also was found, that these extracts has high content of polyphenols and flavonoids.
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Využití symbiotických mikroorganismů a podpůrných nanočástic při zvyšování odolnosti zeleniny v podmínkách vodního stresu
Stašková, Nicole
This thesis deals with the influence of symbiotic microorganisms and supporting nanoparticles on vegetable. Lettuce (Lactuca sativa var. capitata L.), variety "ALAMER" was chosen to be the model vegetable. Symbiotic microorganisms Rhizophagus irregularis and Bacillus subtilis were applied to lettuce plants, and GO-N-1 nanoparticles containing graphene oxide and dicyandiamide were applied to lettuce leaves. The following parameters were evaluated on lettuce plants: height of the above-ground part of plants, weight of the above-ground part of the plants, normalized differentiation index (NDVI), quantum yield of photosynthesis (QY), nitrate content in fresh lettuce leaves and the occurance of mycorrhizical structures.
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Study of effect of Bacillus subtilis yxkO gene on motility during stress response to osmotic upshift.
Streitová, Eliška ; Lichá, Irena (advisor) ; Krásný, Libor (referee)
Bacillus subtilis is gram-positive soil bacteria. In its natural environment it is constantly exposed to changes of chemical and physical conditons, including changes of osmolality. It responds to high osmolality by transporting of potassium ions and afterthat transporting and/or synthetising of compatible solutes. In last years the mutant strain Bacillus subtilis L-42 was isolated with non-specific insertional mutagenesis (mini Tn10) in our laboratory. This strain displays limited growth and inability to cope with hyperosmotic shock in a defined medium with potassium concentration of < 1 mmol/l. Insertion of transposon was located in yxkO gene which encodes a protein of unknown biological function. Some other data also indicate a possible role of disruption of yxkO gene in regulation of expression of hag gene, which encodes flagelin - a pivotal protein of bacterial flagellum. The goal of this thesis was to clarify if the disruption of yxkO gene influences motility and whether is affected the transcription of hag gene. With integrative vector pMUTIN4 a mutant strain with specific mutation of yxkO gene was prepared. Vector was pasted into chromosome of Bacillus subtilis strain 1A839 - genotype of this strain allows to extrude the known transcriptional regulation of hag gene. Cell's motility was...
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Regulation of cell cycle in Bacillus subtilis.
Zelenka, Tomáš ; Lichá, Irena (advisor) ; Harant, Karel (referee)
2 Abstract Relations between several events running in bacterial cell during cell cycle were the subject of many studies during last years. More advanced techniques showed, that bacterial cell life has much more variable factors, than we supposed before. Relatively recent researches managed to reveal function and in few events molecular principle of several mechanisms coordinating those events such as progression of replication and its initiation, segregation of newly replicated chromosomes and after all synchronization of complex cell division machinery. Furthermore it showed variability of those events during changing living conditions of the cell. Keywords: Cell cycle, regulation, initiation, replication, segregation of chromosome, cytokinesis, Bacillus subtilis
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