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Optimalizace produkce vybraných enzymů pomocí Bacillus subtilis
Slavíčková, Radka ; Omelková, Jiřina (oponent) ; Hermanová, Soňa (vedoucí práce)
V diplomové práci byla studována optimalizace produkce lipolytických enzymů při submerzním způsobu kultivace Bacillus subtilis (BS). Produkce lipolytických enzymů byla testovaná v prostředí tří živných médií, která se lišila zejména hlavními zdroji uhlíku, resp. dusíku. První médium obsahovalo zejména extrakt z telecího mozku a hovězího srdce (BHIB), druhé médium pepton a kvasniční extrakt (NB) a třetí živné médium pepton a kvasniční extrakt s přídavkem 2% (w/v) glukózy (NBG). Nejvyšší lipolytické aktivity (0,0784 Uml-1) bylo dosaženo v médiu NBG, přičemž ve všech médiích bylo pozorováno maximum před koncem exponenciální fáze růstu. V NBG médiu bylo následně stanoveno teplotní optimum v rozmezí 30 - 50 °C, pH optimum v rozmezí 5 - 11 a tepelná stabilita lipolytických enzymů produkovaných BS. Aktivita byla stanovena spektrometricky za použití p-nitrofenyllaurátu jako substrátu. Produkované lipolytické enzymy vykazovaly nejvyšší aktivitu při teplotě 37 °C a v alkalické oblasti pH = 8,0. Měření tepelné stability ukázalo, že se jedná o poměrně termostabilní enzymy, které si i po 1 hodině kultivace při 30 - 50 °C zachovaly 100 % aktivity. Přítomnost 1% (w/v) olivového oleje v médiu NBG způsobovala po 14 dnech kultivace pokles lipolytické aktivity o 65 % a pokles hodnoty pH z 6,5 na 5,4. Po nahrazení glukózy fruktózou v živném médiu NBG vykazovala lipolytická aktivita během prvních 7 dní kultivace srovnatelné hodnoty, avšak po 14 dnech kultivace byl zaznamenán již pokles lipolytické aktivity o 29 %. Metodika identifikace lipolytických enzymů BS pomocí peptidového mapování byla vyvinuta za účelem porozumnění potenciálu syntetického polyesteru- poly(e-kaprolaktonu) působit jako induktor tvorby lipáz. Degradační studie komerčního polyesteru poly(e-kaprolaktonu) byla vedena při submerzním způsobu kultivace Bacillus subtilis v médiu NBG při počáteční hodnotě pH 7,0 a teplotě 30 °C po dobu 14 dnů. PCL (Mn=10 000, Mw=14 000) byl studován ve formě filmů (1,0 x 1,0 cm), které byly připraveny lisováním, ochlazováním taveniny na 4 °C a odpařováním dichlormetanu z 2% roztoku. U degradovaných filmů v závislosti na způsobu jejich přípravy byl pozorován po 14 dnech úbytek hmotnosti (7,8 - 17,0 hm. %.) a vznik četných děr a prasklin na povrchu vzorků. V degradačním médiu byly naměřeny hodnoty lipolytické aktivity vyšší o 9 - 17 % ve srovnání s kontrolními vzorky. Denzitometrické sledování nárůstu buněčné hmoty ukázalo rovněž vyšší hodnoty zákalů v degradačním médiu v porovnání s kontrolními vzorky. Na základě naměřených výsledků lze navrhnout, že studované vzorky byly degradovány v důsledku působení BS.
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Microbial Degradation of Polycaprolactone-based Materials
Damborský, Pavel ; Stratilová, Eva (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with the issue of the effect of nutritious factors and aeration on Bacillus subtilis (CCM 1999) lipase production, which was studied from the viewpoint of the crucial action of lipases on polyester chains degradation. The parameters such as bacterial growth, lipolytic activity, pH optimum, temperature optimum, thermal stability, proteolytic activity, the amount of proteins, etc. were determined in three types of media inoculated by Bacillus subtilis. One sample set of media: peptone and yeast extract (NB), medium consisted in peptone and yeast extract with the addition of 2% (w/v) glucose (NBG) and mineral solution with yeast extract (MS-YE) contained poly(-caprolactone), PCL film (Mn = 10 kDa, = 1.4). Experiments were performed for 21 days under shaking at 160 and 200 rpm. The presence of PCL caused in both types of media (NB, NBG) higher values of lipolytic activity of Bacillus subtilis indicating the participation of released low-molecular weight species as the products of PCL biodegradation. In BS-inoculated MS-YE medium low lipolytic activity of Bacillus subtilis as compared with those measured in NB and NBG media was determined even in the presence of PCL. During experiment pH value shifted from neutral (pH 7.0) to alkaline (pH 8.5 – 9.3) region for all types of media with and without PCL specimens. This fact indicates negligible pH changes in microenvironment due to the degradation products and/or metabolites. Lipolytic enzymes determined in supernatants free of bacterial cells have shown two pH optima in the presence of PCL, at pH 7 and 9. Lipase in the absence of polymer displayed optimum pH of 7. Measurement of thermal stability demonstrated that extracellular lipases as relative thermostable enzyme especially in the absence of polymer. The basic proteomic analysis by peptide mass fingerprint (PMF) of lipases produced by Bacillus subtilis at NBG medium after one week of cultivation was performed. The presence of proteins with molecular weight (19.3 kDa) close to that reported for lipases was verified through FPLC. Both SDS-PAGE and IEF-PAGE indicated presence of these proteins in both studied media (NBG vs. NBG/PCL). However, no differences have been found in the presence of PCL and no lipase was truly identified through MALDI-TOF mass spectrometry. The occurrence of degradation process in PCL samples was also documented by their gradual weight loss, which was seen in all media types as the synergic effect of lipase- and base-catalyzed ester bond scission. Model degradation study of PCL and its composite with graphene oxide (2.7 wt% GO) was performed in the presence of Bacillus subtilis in NBG medium at 30 °C and initial pH 7 for three weeks. The weight loss of PCL films gradually increased during whole degradation test up to 12 wt%. Degradation of PCL/GO composite proceeds slower as documented by maximum weight loss of 5.0 wt%. The similar character of elution curves of both PCL and its composite, determined by SEC analysis, indicated shifting to lower molecular region.
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Antimicrobial activity of carbon-based fillers
Stuchlíková, Olga ; Hermanová, Soňa (oponent) ; Voběrková, Stanislava (vedoucí práce)
The aim of this diploma thesis is focused on the impact of carbon-based fillers on viability and extracellular substances production by bacterium Bacillus subtilis (CCM 1999) and yeast Yarrowia lipolytica (CCY 29-26-52). Antimicrobial activity of these particles, present in cultivation nutrient medium was examined using following parameters: growth of mentioned microorganisms, production of extracellular proteins and finally extracellular polymeric substances production, which is strongly connected with biofilm formation. Nanomaterials suspension (0.135 mg/mL) was prepared in two different cultivation media i.e. nutrient medium supplemented with glucose for Bacillus subtilis and basal medium with the addition of 2% (vol.) Tween 80 for Yarrowia lipolytica and media were inoculated by appropriate type of microorganism. Experiments were performed for 6 days under shaking rate at 160 rpm and at temperature of 30 °C for Bacillus subtilis and 28 °C for Yarrowia lipolytica. Three types of carbon nanomaterials obtained from Department of Inorganic Chemistry, Institute of Chemical Technology, Prague were examined. These materials specified as material “A”, “B” and “C” are mutually different by the size of its particles and the degree of oxidation. Based on the screening studies the tested material concentration of 0.135 mg/mL and shaking rate of 160 rpm were chosen. According to the optical density measurement at 600 nm, the growth curves of both microorganisms in the presence of tested nanoparticles during 5 days period were compared. It was find out, that the presence of nanoparticles don’t have a significant influence on tested microorganisms growth, by this technique. However, this method is just wider point of view, due to mistakes caused by presence of dead cells. Further, production of total cells proteins and extracellular proteins by microorganisms in presence of tested nanoparticles was examined. There was not observed any significant deviation from control samples values, where the tested materials were absent. Based on colony counting method (used for Bacillus subtilis) and cells counting in Bürker counting chamber (used for Yarrowia lipolytica), loss of microorganism viability was determined in 3 cultivation periods (6, 48 and 144 hours); there was observed a support of growth of microorganisms rather in shorter incubation period. Thereafter the extracellular polymeric substances (EPS) production that means proteins, reducing substances and polysaccharides was monitored. These substances were secreted into the medium by mentioned microorganisms during 24 hours of incubation. Bacillus subtilis cells produce much more EPS than Yarrowia lipolytica cells. We suppose that the EPS production could be closely associated with production of biofilm, which protects cells against nanoparticles toxicity.
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Biocatalysts based on lipases, their immobilization and characterization
Bančáková, Anna ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with the issue of immobilized enzymes. In the theoretical part, principal and novel methods of immobilization and types of carriers are reviewed together with practical applications of immobilized enzymes. Particularly, recent utilization of immobilized enzymes in a variety of industries such as food industry, medicine, chemical analysis, bioremediation, and biodiesel production is summarized. Different methodologies including adsorption, entrapment, covalent binding and cross-linking are discussed from the viewpoint of their dis/advantages resulting from the extent and the character of binding and the maintenance of enzyme activity. Among new carriers, the research work done on the utilization of graphene as a novel carrier for enzyme immobilization is particularly reported. In the experimental part, immobilization of commercial preparation of lipase (RA) isolated from microscopic fungi Rhizopus arrhizus was performed by adsorption on polyethylene terephthalate as a carrier. The basic parameters as lipolytic activity, temperature optimum, pH optimum and thermal stability of both soluble as well as immobilized enzyme were determined spectrophotometrically using p-nitrophenyl-laurate (pNPL) as a substrate. Both the soluble and immobilized lipase showed maximum activity at pH 7.2 and a decrease in activity was observed above pH 8 or below pH 6.5. The dependence of activity on pH of reaction medium was more pronounced in the case of immobilized form of enzyme. The soluble and immobilized lipases exhibited maximum activity at a temperature of approximately 30 °C. A drop in activity values was observed when the temperature was increased up to 50 °C and above this temperature the stability of the soluble lipase sharply decreased. On the contrary, thermal stability of immobilized RA lipase was significantly improved. Immobilized form of enzyme possesses activity 3.7 % in comparison to the soluble form.
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Studium mechanizmu a kinetiky koordinační polymerace hexa-1,5-dienu katalyzované fenoxyiminovým komplexem titanu a MAO
Ševčík, Jan ; Cihlář, Jaroslav (oponent) ; Hermanová, Soňa (vedoucí práce)
Tato diplomová práce se zabývá studiem polymerace hexa-1,5-dienu katalyzované fenoxyiminovým komplexem titanu (FI Ti) a methylaluminoxanem (MAO) jako kokatalyzátorem. Byl studován vliv koncentrace monomeru, polymerační teploty a poměru kokatalyzátor/katalyzátor na aktivitu systému, polydisperzitu a v některých případech i na strukturu připraveného poly(hexa-1,5-dienu). 1H NMR spektroskopie prokázala přítomnost methylen-1,3-cyklopentanových (MCP) a vinyl tetramethylenových (VTM) jednotek v řetězci poly(hexa-1,5-dienu). Byla také studována kinetika této polymerace. V poslední části diplomové práce byl připraven kopolymer ethenu s hexa-1,5-dienem se zabudovanými postranními vinylovými skupinami.
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Katalytické systémy pro polymerace cyklických esterů
Krpoun, Karel ; Hermanová, Soňa (oponent) ; Richtera, Lukáš (vedoucí práce)
Jednou z významných oblastí výzkumu organokovových sloučenin lanthanoidů, které je v poslední dekádě věnována pozornost, je využití komplexů v homogenní katalýze pro syntézu funkčních materiálů s definovanými vlastnostmi např. pro polymerace za otevření kruhu (ring opening). Polymerací za otevření kruhu mohou být připraveny alifatické polyestery, které díky své biodegradabilitě a biokompatibilitě nachází mimo jiné uplatnění i v biomedicíně. Teoretická část předložené práce se zabývá přípravou lathanidocenů, jejich charakterizací a možnostmi jejich využití při syntéze polymerů. Cílem experimentální části bylo provedení a optimalizace syntézy série lanthanocenových chloridových komplexů (Sm, Nd a Pr) a monopentamethylcyklopentadienylových komplexů. Uvedené strukturní typy byly vybrány s ohledem na možnost polymerační aktivity nebo možnosti jejich aktivace přidáním alkylačních činidel. Komplexy byly spektrálně charakterizovány pomocí NMR a IR spektroskopie a byla určena jejich struktura pomoci rentgenové strukturní analýzy. U vybraných chloridových komplexu neodymu a samaria komplexů byly provedeny studie katalytické aktivity pro polymerace ?-caprolactonu za laboratorní teploty v toluenu. Polykarpolakton byl poté charakterizován pomoci gelové permeační chromatografie a NMR.
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Antimicrobial activity of carbon-based fillers
Stuchlíková, Olga ; Hermanová, Soňa (oponent) ; Voběrková, Stanislava (vedoucí práce)
The aim of this diploma thesis is focused on the impact of carbon-based fillers on viability and extracellular substances production by bacterium Bacillus subtilis (CCM 1999) and yeast Yarrowia lipolytica (CCY 29-26-52). Antimicrobial activity of these particles, present in cultivation nutrient medium was examined using following parameters: growth of mentioned microorganisms, production of extracellular proteins and finally extracellular polymeric substances production, which is strongly connected with biofilm formation. Nanomaterials suspension (0.135 mg/mL) was prepared in two different cultivation media i.e. nutrient medium supplemented with glucose for Bacillus subtilis and basal medium with the addition of 2% (vol.) Tween 80 for Yarrowia lipolytica and media were inoculated by appropriate type of microorganism. Experiments were performed for 6 days under shaking rate at 160 rpm and at temperature of 30 °C for Bacillus subtilis and 28 °C for Yarrowia lipolytica. Three types of carbon nanomaterials obtained from Department of Inorganic Chemistry, Institute of Chemical Technology, Prague were examined. These materials specified as material “A”, “B” and “C” are mutually different by the size of its particles and the degree of oxidation. Based on the screening studies the tested material concentration of 0.135 mg/mL and shaking rate of 160 rpm were chosen. According to the optical density measurement at 600 nm, the growth curves of both microorganisms in the presence of tested nanoparticles during 5 days period were compared. It was find out, that the presence of nanoparticles don’t have a significant influence on tested microorganisms growth, by this technique. However, this method is just wider point of view, due to mistakes caused by presence of dead cells. Further, production of total cells proteins and extracellular proteins by microorganisms in presence of tested nanoparticles was examined. There was not observed any significant deviation from control samples values, where the tested materials were absent. Based on colony counting method (used for Bacillus subtilis) and cells counting in Bürker counting chamber (used for Yarrowia lipolytica), loss of microorganism viability was determined in 3 cultivation periods (6, 48 and 144 hours); there was observed a support of growth of microorganisms rather in shorter incubation period. Thereafter the extracellular polymeric substances (EPS) production that means proteins, reducing substances and polysaccharides was monitored. These substances were secreted into the medium by mentioned microorganisms during 24 hours of incubation. Bacillus subtilis cells produce much more EPS than Yarrowia lipolytica cells. We suppose that the EPS production could be closely associated with production of biofilm, which protects cells against nanoparticles toxicity.
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Study on interactions between lipase and carbon-based support
Hamrlová, Romana ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with issue of immobilized lipase, which was studied concretely from the aspect of interactions between lipase and carbon-based carrier. Immobilization of lipase from Rhizopus arrhizus (RA) was performed by adsorption onto graphene oxide types of carrier (a1, a, b, c and d) and by covalent attachment onto poly(ethylene glycol) modified carrier a1, PEG-a1. Adsorbed enzyme was additionally cross-linked with glutaraldehyde, GA(RA-a1). The influence of hydrophobic character of the carrier surface was confirmed since enzyme immobilized onto carriers with more hydrophobic surface (less polar groups) achieved higher activity retention at raising enzyme concentration. All activity assays were done spectrophotometrically using p-nitrophenyl laurate (p-NPL) as a substrate. The basic biochemical and kinetics characteristics were determined for immobilized as well as soluble enzyme. The pH optimum of covalently attached and adsorbed enzyme was shifted to more acidic environment (pH 7-8), whereas for soluble enzyme the optimum was achieved at pH 9. The thermostability of immobilized samples was significantly improved in the case of GA(RA-a1), where the glutaraldehyde chemistry was additionally involved. The glutaraldehyde cross-linking of adsorbed enzyme lead to the enhancement of the thermostability, which may be the consequence of intermolecular covalent linkage. The storage stability evaluation showed great improvement of lipolytic activity retention during the condition of the samples at 4 °C in phosphate buffer. Soluble enzyme lost more than 84 % of its initial activity after 42 days, while the immobilized enzyme onto c carrier kept still 100 % of its initial activity. The best storage stability showed the c carrier while after 180 days still kept 87 % of its initial activity.
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Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (oponent) ; Hermanová, Soňa (vedoucí práce)
The diploma thesis deals with the issue of the effect of tweens on enzymatic activity of model hydrolase both free and immobilized on carbon-based carrier. In theoretical part, structural features, mechanism of action, and specialty applications of microbial lipases are reviewed along with detergent chemistry, with emphasize on tween family of detergents belonging into non-ionic surfactant group. In experimental part, effect of tweens on soluble as well as immobilized hydrolase was examined. Immobilization of commercial preparation of lipase was performed by non-covalent adsorption on graphene oxide as a carrier treated with different tweens (tween 20, 60, 80). The activity was determined spectrophotometrically by p-nitrophenyl laurate assay. Enhancement of soluble Rhizopus arrhizus lipase activity (activity coupling of 104 %) was observed at tween 20 concentration of 10 mmol•dm-3, which is highly above critical micelle concentration of this detergent. On the base of screening study, immobilization protocol comprised the incubation of soluble enzyme at concentration of 0.1 mg•ml-1 in phosphate buffer (pH 7.2) with tween 20 (10.8 mmol•dm-3) and the carrier for one hour. Both soluble and immobilized lipase exhibited maximum activity at approx. 35 °C. Optimal pH of immobilized lipase shifted to 8 compared to soluble form for which pH optimum at 9 was determined. Thermal stability profile follows almost same trend for both soluble and immobilized enzyme samples. The interactions between carrier and enzyme are suggested to be mainly non–covalent (adsorption, electrostatic interactions). No protein leaching was observed under studied conditions, and significant improvement of storage stability of immobilized lipase was achieved (activity retention of 41 % after 110 days) in comparison with soluble lipase (activity retention of 16 % after 42 days).
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Microbial Degradation of Polycaprolactone-based Materials
Damborský, Pavel ; Stratilová, Eva (oponent) ; Hermanová, Soňa (vedoucí práce)
The thesis deals with the issue of the effect of nutritious factors and aeration on Bacillus subtilis (CCM 1999) lipase production, which was studied from the viewpoint of the crucial action of lipases on polyester chains degradation. The parameters such as bacterial growth, lipolytic activity, pH optimum, temperature optimum, thermal stability, proteolytic activity, the amount of proteins, etc. were determined in three types of media inoculated by Bacillus subtilis. One sample set of media: peptone and yeast extract (NB), medium consisted in peptone and yeast extract with the addition of 2% (w/v) glucose (NBG) and mineral solution with yeast extract (MS-YE) contained poly(-caprolactone), PCL film (Mn = 10 kDa, = 1.4). Experiments were performed for 21 days under shaking at 160 and 200 rpm. The presence of PCL caused in both types of media (NB, NBG) higher values of lipolytic activity of Bacillus subtilis indicating the participation of released low-molecular weight species as the products of PCL biodegradation. In BS-inoculated MS-YE medium low lipolytic activity of Bacillus subtilis as compared with those measured in NB and NBG media was determined even in the presence of PCL. During experiment pH value shifted from neutral (pH 7.0) to alkaline (pH 8.5 – 9.3) region for all types of media with and without PCL specimens. This fact indicates negligible pH changes in microenvironment due to the degradation products and/or metabolites. Lipolytic enzymes determined in supernatants free of bacterial cells have shown two pH optima in the presence of PCL, at pH 7 and 9. Lipase in the absence of polymer displayed optimum pH of 7. Measurement of thermal stability demonstrated that extracellular lipases as relative thermostable enzyme especially in the absence of polymer. The basic proteomic analysis by peptide mass fingerprint (PMF) of lipases produced by Bacillus subtilis at NBG medium after one week of cultivation was performed. The presence of proteins with molecular weight (19.3 kDa) close to that reported for lipases was verified through FPLC. Both SDS-PAGE and IEF-PAGE indicated presence of these proteins in both studied media (NBG vs. NBG/PCL). However, no differences have been found in the presence of PCL and no lipase was truly identified through MALDI-TOF mass spectrometry. The occurrence of degradation process in PCL samples was also documented by their gradual weight loss, which was seen in all media types as the synergic effect of lipase- and base-catalyzed ester bond scission. Model degradation study of PCL and its composite with graphene oxide (2.7 wt% GO) was performed in the presence of Bacillus subtilis in NBG medium at 30 °C and initial pH 7 for three weeks. The weight loss of PCL films gradually increased during whole degradation test up to 12 wt%. Degradation of PCL/GO composite proceeds slower as documented by maximum weight loss of 5.0 wt%. The similar character of elution curves of both PCL and its composite, determined by SEC analysis, indicated shifting to lower molecular region.
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