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Hydrogels with incorporated enzymes
Geistová, Karolína ; Krouská, Jitka (referee) ; Mravec, Filip (advisor)
This bachelor thesis deals with the study of incorporation of enzymes into phase separated hydrogels. The aim of this work is to determine the enzyme activity in phase separated gels. Gels were prepared by the dry-way based on the interaction of negatively charged polyelectrolyte (hyaluronan) with positively charged surfactant (Septonex). Two enzymes, bromelain and collagenase, were incorporated into the hydrogels. To determine enzyme activity, the modified albumin protein with bound sulfanilamide group (azoalbumin) was used as a substrate. The enzyme activity of the enzyme itself, the enzyme activity affected by one of the two components of the system as well as the activity of the enzyme directly in the hydrogel was determined on UV-VIS spectrophotometry. The enzyme was found to be incorporated in the hydrogel. Furthermore, a significant effect of the positively charged surfactant on the enzyme activity was detected in phase-separated hydrogels.
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Thermophilic Enzymes
Kuchtová, Ludmila ; Obruča, Stanislav (referee) ; Babák, Libor (advisor)
Thermophilic enzymes are produced by organisms living at temperatures between 60 and 80°C, in some cases even higher, and are active and stabile at these temperatures. This work deals with thermophilic and hyperthermophilic organisms, their habitats, ways of adaptation to environment with considerably higher temperatures than optimal temperature for most organisms, and recent findings and opinions about possible factors enhancing protein thermostability. Properties and behaviour of thermophilic enzymes and their current applications (i.e. PCR, starch industry) are summarized. Advantages and disadvantages of using of these enzymes in comparison to currently used mesophilic enzymes are compared. Possible applications of thermozymes in various industrial branches, where they could replace some environment polluting procedures, simplify technology and be economically important, are introduced.
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Isolation, characterization and application of biomedically important polymer P(3HB-co-4HB)
Krupičková, Kristýna ; Přikryl, Radek (referee) ; Obruča, Stanislav (advisor)
This diploma thesis deals with the isolation and characterization of copolymer P(3HB-co-4HB). The teoretical part was prepared as a literature search which describe polyhydroxyalkanoates in general, their structure, synthesis, degradation and isolation. Furthermore, copolymers containing 4HB units are mentioned in this thesis and there is also no mentioned of the biosynthesis and biodegradation of copolymer P(3HB-co-4HB). The first aim of this diploma thesis was to find out which solvent is the best for copolymer extraction and then characterize obtained copolymer P(3HB-co-4HB). The isolated copolymer was characterized by gas chromatography, SEC-MALS, thermal analysis and SEM. In the second part of the thesis, release of model biologically active substance from the PHA films was studied. Rhodamine 6G dye was selected for the simulation, which was used to stain the copolymer films and the P(3HB) films. It was observed that film prepared from P(3HB-co-4HB) copolymer released entrapped substance much faster than film made from P3HB homopolymer, and, in addition, the copolymer was substantially more susceptible to enzyme degradation.
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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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Study on protective effect of 3-hydroxybutyrate and its derivatives
Černá, Klára ; Smilek, Jiří (referee) ; Obruča, Stanislav (advisor)
The aim of this thesis is the study of chemical chaperones, as a preservative enzymes before denaturing processes. In the experimental part of the study we investigated the protective effects of five selected potential protective compounds – derivatives of 3-hydroxybutyrate – two model enzymes: lipase and lysozyme. Protective effects of potential chemical chaperones were investigated by dynamic light scattering (DLS), which was determined by the size distribution of the aggregates lipase produced depending on the temperature at the heat-induced denaturation. Further we have been used differential scanning calorimetry (DSC), which was determined by the temperature of the denaturation of lysozyme in the presence or absence of potentially protective substances. The last parameter was to determine the effect of potentially protective agents at different concentrations on the catalytic activity of lipase and determining the residual lipase activity after application of denaturing two factors – the high temperature and freezing. Of all tested structural analogues of 3-hydroxybutyrate was the highest protective effect observed (stabilization of enzyme molecules) with the succinate, which has two carboxyl groups. Conversely, 1,3-butanediol showed virtually no protective activity, indicating that the presence of carboxyl groups on the protective effect of essential. The low protective efficacy butyrate further indicates that it is essential that an effective protectant in addition to the carboxyl group contained as a further functional group – either carboxyl or hydroxyl. More significant protective effect was observed in 3-hydroxybutyrate than in 2-hydroxybutyrate. Interestingly, the effective protectant while at higher concentrations strongly inhibit the enzyme activity of the model enzyme, which is probably related to the solvation and conformations of the protein in the presence of protectant and the availability of the active site for the substrate.
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New fungal fucosidases and their synthetic potential
Glozlová, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Glozlová Supervisor: prof. PharmDr. Martin Beránek, Ph.D. Consultants: doc. RNDr. Pavla Bojarová, Ph.D., Mgr. Pavlína Nekvasilová Title of diploma thesis: New fungal fucosidases and their synthetic potential The literary research focuses on α-L-fucosidases, their occurrence, functions, and use in the synthesis of fucosylated compounds, which were also a part of the research, particularly their presence in the human body and involvement in pathological conditions. Furthermore, it explores the oligosaccharides found in human milk, their impact on infant health, and their synthesis, which can be facilitated through the use of α-L-fucosidases. The experimental section involves the analysis of a library of filamentous fungi with the aim of identifying a representative strain with α-L-fucosidase activity towards pNP-α-L-fucoside. Recombinant α-L-fucosidases were selected based on searches in genetic databases and prepared. The feasibility of their production in Pichia pastoris and Escherichia coli was tested, and the produced enzymes were purified by ion exchange chromatography, characterized and their regioselectivity and synthetic potential for the synthesis of fucosylated...
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Effect of enzymes on the degradation of thermosensitive copolymers for controlled release of drugs
Dávidíková, Anna Mária ; Michlovská, Lenka (referee) ; Vojtová, Lucy (advisor)
Termocitlivé hydrogély, ktoré sa používajú ako nosiče liečiv v dnešnej dobe zažívajú obrovský záujem v spoločnosti. Používajú sa ako spôsob dopravy liečiva na predom určené miesto a po dosiahnutí určeného miesta slúžia ako dlhodobé úložisko liečiv v tele. Vlastnosti hydrogélov môžu byť upravované tak, aby dokázali naviazať rôzne druhy liečiv, aby boli citlivé na rôzne vonkajšie impulzy a aby degradovali pomalšie, čo predĺži životnosť liečiv v tele. Aby sme vedeli, ako upraviť vlastnosti hydrogélov a ako sa budú správať, musíme poznať ich degradačný proces a chovanie v rôznych degradačných médiách. Práve preto sú degradačné štúdie veľmi dôležitou súčasťou štúdií hydrogélov. Táto bakalárska práca je zameraná na sledovanie degradácie rôznych roztokov termocitlivého hydrogélu vo viacerých degradačných médiách. Teoretická časť práce zahrňuje krátky prehľad o tom, čo sú to termocitlivé hydrogély, ich charakterizácia a popis vlastností spolu s použitím hydrogélov ako nosičov liečiv. Ďalej sa teoretická časť venuje degradácií hydrogélov celkovo ako aj degradácií konkrétne použitého hydrogélu v tejto bakalárskej práci. V prvom rade bol syntetizovaný živou polymeráciou za otváraním kruhu v inertnej atmosfére termocitlivý PLGA-PEG-PLGA kopolymér, tvorený poly(mliečnou-co-glykolovou kyselinou)-b-poly(ethylen glykolom)-b-poly(mliečnou-co-glykolovou kyselinou). Následne bol kopolymér charakterizovaný na protónovej nukleárnej magnetickej rezonancii a na gélovej chromatografii. Z nasyntetizovaného kopolyméru boli pripravené tri vodné roztoky hydrogélu o rôznych koncentráciách a to 10, 15 a 20 hm %. Vzorky pripravených hydrogélov za teploty 37 , boli degradované v rôznych degradačných roztokoch (UPW (ultra čistá voda), NaCl, PBS (fosfátový pufr) a roztok PBS s enzýmom). Degradačná štúdia bola hlavným cieľom tejto bakalárskej práce. Jednotlivé vzorky boli odobrané v konkrétne stanovené časy a to po 1, 2, 3, 7, 9, 14, 16, 21, 23 a 30 dňoch. Pri vzorkách s enzýmom bola predpokladaná rýchlejšia degradácia vzoriek, tak tieto odbery boli nastavené po 1, 2 a 3 dňoch. Ďalším cieľom tejto práce bola analýza odobraných vzoriek hydrogélov po degradácií na gélovej chromatografii a analýza degradačných médií na kvapalinovej chromatografií s hmotnostným spektrometrom. Počas degradácie vzoriek bola prevedená taktiež gravimetrická analýza a pri odoberaní degradačných roztokov bolo merané ich pH. Následne boli všetky získané dáta vyhodnotené a bolo sledované, či enzým urýchľuje PLGA-PEG-PLGA hydrolytickú degradačnú kinetiku. Výsledky poukazujú na to, že enzým má nízky vplyv na rýchlosť degradácie. Výsledky tejto práce poslúžia ako základy pre budúce degradačné štúdie.
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New fungal fucosidases and their synthetic potential
Glozlová, Michaela ; Beránek, Martin (advisor) ; Matoušková, Petra (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Bc. Michaela Glozlová Supervisor: prof. PharmDr. Martin Beránek, Ph.D. Consultants: doc. RNDr. Pavla Bojarová, Ph.D., Mgr. Pavlína Nekvasilová Title of diploma thesis: New fungal fucosidases and their synthetic potential The literary research focuses on α-L-fucosidases, their occurrence, functions, and use in the synthesis of fucosylated compounds, which were also a part of the research, particularly their presence in the human body and involvement in pathological conditions. Furthermore, it explores the oligosaccharides found in human milk, their impact on infant health, and their synthesis, which can be facilitated through the use of α-L-fucosidases. The experimental section involves the analysis of a library of filamentous fungi with the aim of identifying a representative strain with α-L-fucosidase activity towards pNP-α-L-fucoside. Recombinant α-L-fucosidases were selected based on searches in genetic databases and prepared. The feasibility of their production in Pichia pastoris and Escherichia coli was tested, and the produced enzymes were purified by ion exchange chromatography, characterized and their regioselectivity and synthetic potential for the synthesis of fucosylated...
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The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase
Sobolová, Zuzana ; Mladěnka, Přemysl (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Sobolová Supervisor: Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Consultant: Marcel Hrubša, MS.c. Title of diploma thesis: The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase Inhibitors of alcohol dehydrogenase play a key role in the treatment of poisoning caused by methanol, ethanol, ethylene glycol, and their toxic metabolites. Such treatment can therefore be considered as life-saving. In clinical practice, a single substance, fomepizole, is used. This thesis is aimed at investigation of the effect of eleven experimental chelators from the 4- acylpyrazol-5-ones group on equine and yeast alcohol dehydrogenases, and the possible definition of structural features that would be the most suitable for interaction with this enzyme. Due to the ability of these substances to chelate zinc ions, it is believed that they could inhibit an enzyme that has a zinc molecule in the structure. The measurement itself is based on principle of increase in absorbance during the enzymatic reaction, which is caused by the formation of NADH. This measurement is relatively simple, efficient and, due to used kinetic method, also less susceptible to be affected by...
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