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Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (referee) ; Hermanová, Soňa (advisor)
Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.
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Utilization of light scattering techniques in study on protein denaturation
Köbölová, Klaudia ; Kalina, Michal (referee) ; Sedláček, Petr (advisor)
This bachelor thesis is focused on verifying the use of light scattering techniques in the study of protein denaturation. The theoretical part of the thesis describes proteins, their general properties, the process of denaturation and it provides an insight into light scattering. In the experimental part of the study I investigated the denaturation effects of four selected potential denaturing agents – urea, guanidine hydrochloride, sodium dodecyl sulfate, copper (II) sulfate – on two model enzymes: lipase and lysosyme. The denaturation effects of these denaturants were studied with the help of the dynamic light scattering method (DLS). In order to describe the change in particle size during denaturation, I used Z-Average, Volume mean and intensity distribution which were determined by this method.
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Biocatalysts based on lipases, their immobilization and characterization
Bančáková, Anna ; Voběrková, Stanislava (referee) ; Hermanová, Soňa (advisor)
Bakalárska práca sa zaoberá problematikou imobilizovaných enzýmov. V teoretickej časti sú zhrnuté najnovšie používané metódy imobilizácie, typy heterogénnych nosičov a taktiež praktické využitie imobilizovaných enzýmov v potravinárskom priemysle, medicíne, chemickej analýze, pri produkcii bionafty a bioremediácii. Uvedené sú tiež výhody a nevýhody jednotlivých metód imobilizácie (adsorpcia, zachytenie, kovalentné spojenie a zasieťovanie) plynúce z charakteru spojenia enzýmu a nosiča a výslednej aktivity imobilizovaného enzýmu. Z hľadiska nových typov heterogénnych nosičov práca konkrétne uvádza využitie grafénu, ktorý je pre svoje špecifické fyzikálno-chemické vlastnosti často používaným a skúmaným nosičom v posledných rokoch. V experimentálnej časti bola prevedená imobilizácia komerčného preparátu lipázy (RA), izolovanej z mikroskopickej plesne Rhizopus arrhizus, adsorpciou na polyetyléntereftalát, ktorý bol použitý ako nosič. U voľnej i imobilizovanej formy enzýmu boli stanovené základné parametre ako lipolytická aktivita, teplotné optimum, pH optimum a tepelná stabilita. Aktivita enzýmu bola meraná spektrofotometricky pri vlnovej dĺžke 420 nm. Ako substrát bol použitý roztok p-nitrofenyl-laurátu. U voľného aj imobilizovaného enzýmu bolo stanovené pH optimum 7,2, pokles aktivity bol zaznamenaný pri pH nad 8 a pod 6,5. Väčšia závislosť výslednej aktivity na pH prostredia bola dokázaná u imobilizovanej formy enzýmu. U voľného aj imobilizovaného enzýmu bola pozorovaná najvyššia aktivita pri teplote 30 °C. Pokles aktivity bol pozorovaný po zvýšení teploty nad 50 °C. Po dosiahnutí tejto teploty stabilita rozpustného enzýmu prudko klesala. Avšak tepelná stabilita skúmanej lipázy sa po jej imobilizácii výrazne zlepšila. Imobilizovaná forma enzýmu mala v porovnaní s voľnou formou aktivitu 3,7 %.
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Stanovení aktivity průmyslově významných enzymů produkovaných mikroorganismy izolovanými z potravinového odpadu
Lymarenko, Sofiia
The microbiome and the produced enzymes play an important role in processing food waste. Bacillus licheniformis is one of the representatives of microflora in composting and producer of industrially important hydrolytic enzymes: protease, lipase and cellulase. As part of the thesis, enzymatic activity using spectrophotometric methods was determined in seven strains of B. licheniformis for a period of 96 to 192 hours. In all seven strains, cellulase, protease and lipase activity was detected, which agrees with previously published literature. The highest cellulase activity determined by two methods was recorded for strain 3A-52 (1 U/ml) and 1A-52 (1.5 U/ml) after 24 hours of cultivation. The pH optimum for cellulase activity was 7, and the temperature optimum 50 °C to 60 °C. The maximum protease activity was detected after 120 and 144 hours of cultivation in two strains: 3A-52 (1.3 U/ml) and 3D-42 (1.4 U/ml). During the optimization, it was found that the pH optimum was 9 to 11 and the temperature optimum was 40 to 45 °C depending on the tested strain. The maximum lipase activity was approximately 0.7 U/ml after 144 hours of cultivation in 4D-41 and 3D-42. The temperature optimum was found at 50 to 60 °C and the range of pH optimum for this activity was very wide depending on the studied strain. For the strains with the highest enzymatic activity, a growth curve was determined, where the highest cellulase activity corresponded to the time of transition of the bacterial culture from the exponential phase to the stationary phase, and the highest lipase and protease activities were found later only at the transition from the stationary phase to the death phase. Strain 3A-52 and 3D-42 showed the highest activity for all three monitored enzymes. Subsequently, these strains isolated during composting can be used to produce enzymes industrially.
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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Utilization of light scattering techniques in study on protein denaturation
Köbölová, Klaudia ; Kalina, Michal (referee) ; Sedláček, Petr (advisor)
This bachelor thesis is focused on verifying the use of light scattering techniques in the study of protein denaturation. The theoretical part of the thesis describes proteins, their general properties, the process of denaturation and it provides an insight into light scattering. In the experimental part of the study I investigated the denaturation effects of four selected potential denaturing agents – urea, guanidine hydrochloride, sodium dodecyl sulfate, copper (II) sulfate – on two model enzymes: lipase and lysosyme. The denaturation effects of these denaturants were studied with the help of the dynamic light scattering method (DLS). In order to describe the change in particle size during denaturation, I used Z-Average, Volume mean and intensity distribution which were determined by this method.
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Enzymatic modification of Glc-NAc-thiazoline inhibitors of hexosaminidase
Šmeringaiová, Ingrida ; Weignerová, Lenka (advisor) ; Šulc, Miroslav (referee)
This work deals with the problem of searching for effective derivatives of 1,2-dideoxy-2'- methyl-α-D-glucopyranoso-[2,1-d]-Δ2'-thiazoline (NGT), potential inhibitors of human β-N- acetylhexosaminidases. The work is targeted to production of inhibitors derived from NGT by the modification of its structure by free and immobilised lipases. Potential inhibitors of β-N- acetylhexosaminidases may be potent tools for studying of enzymes in cell processes and also open possibility to the discovery of new possible drugs for the treatment of some neurodegenerative diseases, such as Alzheimer disease. The thesis is focused on brief characterization of β-N-acetylhexosaminidases, e.g., glycosidases from enzymatic groups GH 20 and GH 84. β-N-Acetylhexosaminidases from microorganisms Bacteroides thetaiotaomicron, Aspergillus oryzae, Streptomyces plicatus, Talaromyces flavus and humans1 were used in the experiments. The enzymes produced were purified and then tested for their activity. We also tested inhibitory activity of potential inhibitor 6-O-acetyl-1,2-dideoxy-2'-methyl-α- D-glucopyranoso-[2,1-d]-Δ2'-thiazoline. Starting compound, NGT, was synthesized by the modified process (originally created by prof. Knapp2 ) using 2-acetamido-1,3,4,6-tetra-O- acetyl-2-deoxy-α-D-glucopyranose in the reaction with...
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Glycocalyx shedding by cercariae of bird schistosomes
Chaloupecká, Jana ; Mikeš, Libor (advisor) ; Štěrba, Ján (referee)
Trichobilharzia spp. are avian schistosomes related to medically important human parasites of the genus Schistosoma. Penetrating cercariae are well known as causative agent of cercarial dermatitis in humans. Cercariae actively penetrate the skin of definitive hosts and transform into schistosomula. This process is preceded by cercarial tail detachment and includes emptying of penetration glands and extensive surface changes. One of these changes is the loss of highly immunogenic glycocalyx which represents a protective coat in the aquatic environment. The glycocalyx has specific composition of saccharide molecules which are bound to lipids or proteins on the membrane of cercarial tegument. There is only limited information about the mechanism of shedding. Hypotheses based on indirect evidences suggest that peptidases or (phospho)lipases from penetration glands could be involved. This work describes the changes in surface glycosylation during transformation of cercariae into schistosomula by fluorescently labelled lectins and monoclonal antibodies against Lewis X antigen. Lectins UEA-I, LTA and PNA have been chosen as markers of transformation of T. regenti. Further, our experiments have been focused on shedding of cercarial glycocalyx. During in vitro induction of penetration gland emptying and...
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Effects of detergents on activity, thermostability and aggregation of immobilized lipases
Bančáková, Anna ; Voběrková, Stanislava (referee) ; Hermanová, Soňa (advisor)
Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.
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