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.

Tématem diplomové práce je imobilizace lipáz a konkrétně studium interakcí mezi lipázou a nosičem na bázi uhlíku. Lipáza izolovaná z kmene Rhizopus arrhizus byla adsorbována přímo na různé typy grafen oxidu (a1, a, b, c a d) a na grafen oxid typu a1 modifikovaný poly(ethylenglykolem), PEG-a1. Enzym adsorbovaný na nosič a1 byl následně sesítěn pomocí glutaraldehydu a vzorek byl označen jako GA(RA-a1). Vliv hydrofobního charakteru povrchu nosiče na účinnost imobilizace byl potvrzen vyšší úchovou počáteční aktivity enzymu imobilizovaného na více hydrofobním nosiči (nižší koncentrace polárních skupin) i při vyšší koncentraci rozpustného enzymu v roztoku. Stanovení enzymové aktivity bylo provedeno spektrofotometricky za použití p-nitrofenyl laurátu (p-NPL) jako substrátu. Pro imobilizovaný a volný enzym byly stanoveny základní biochemické a kinetické parametry. Optimální pH kovalentně imobilizovaného enzymu bylo posunuto do více kyselého oblasti (pH 7-8) ve srovnání s volným enzymem, kdy bylo optimum dosaženo při pH 9. Tepelná stabilita imobilizovaného enzymu byla výrazně zlepšena v případě vzorku GA(RA-a1), kde bylo aplikován glutaraldehyd po adsorpci enzymu na nosič. Sesíťování adsorbovaného enzymu pomocí glutaraldehydu vedlo ke zlepšení tepelné stability vzorku, a to pravděpodobně v důsledku intermolekulárních kovalentních vazeb. Na základě měření stability enzymu při teplotě 4 C ve fosfátovém pufru bylo prokázáno značné zlepšení úchovy lipolytické aktivity imobilizovaného vzorku oproti volnému enzymu. Volný enzym ztratil více než 84 % své původní aktivity za 42 dní, zatímco imobilizovaný enzym na nosiči c si zachoval 100 % své původní aktivity. Nejlepší stabilitu enzymu při úchově měl nosič c, když si po 180 dnech stále zachoval 87 % své původní aktivity.

The theoretical part of this thesis deals with cellulolytic enzymes, their microbial producers, the possibilities of using such enzymes in the industry and how can be enzymes - not only cellulolytic - immobilized. Experimental part examines the preparations created by immobilizing various amounts of the commercially used cellulolytic complex Cellulast 1.5L onto various synthetic carriers made of polyethylene terephthalate - commercially used Sorsilen, PET carrier and glutaraldehyde-treated PET carrier. Enzyme activity of these preparations was determined by Somogyi - Nelson method by spectrophotometry. For the highest activity immobilized preparation was determined the temperature- and the pH-optimum. The difference in effects change between the free and immobilized enzyme by measuring viscosity decrease of the substrate depending on the degradation of glycosidic bonds was also studied.

In this thesis the use of waste materials for the microbial production of important metabolites is reported. The first part is focused on the use of waste paper (a lignocellulosic material) as a non-traditional source for the production of bioethanol. The second part is focused on the immobilization of cellulolytic enzymes, which are used for the hydrolysis of lignocellulosic materials. First, the waste paper (cardboard) was pre-treated using a blender and a vibratory mill. The pre-treated cardboard was used for the production of ethanol by the method of simultaneous saccharification and fermentation. This method was optimized with free cells of Saccharomyces cerevisiae. Then strains suitable for the immobilization were selected. Strains of S. cerevisiae and Pichia kudriavzevii were immobilized by encapsulation into the polyvinyl alcohol carrier and tested again for the ethanol production by simultaneous saccharification and fermentation. In the second part of the work a carrier from waste polyethylene terephthalate bottles was prepared and used for the immobilization of the cellulolytic complex. The basic characteristics were determined, such as optimal pH and optimal temperature, storage, operational and thermal stability, enzyme kinetics and the mode of action of the enzyme. Compared to two other commercial carriers this carrier showed to be suitable for the immobilization of the cellulolytic complex.

Biocatalytic membranes (BMs) have promising applications in a diversity of fields including food, pharmaceutical and water treatment industries. Of particular relevance, Alcalase is a commercially important protease that has been applied for the production of peptides from the hydrolysis of proteins. In this study, two different approaches were applied for the modification of electrospun polyacrylonitrile nanofibrous membranes (EPNMs) for Alcalase immobilization. The first approach is alkali modification of EPNMs followed by EDC/NHS coupling for covalent bonding with Alcalase, whereas the other is based on polydopamine coating with or without glutaraldehyde grafting as a covalent linker. Immobilized Alcalase on these prepared BMs were studied and compared with free enzymes. It was found that the stabilities of Alcalase on BMs created using both approaches were improved, which enabled their reuse of 10 cycles with significant retention of enzymatic activity. A continuous reactor housing BMs were tested for hydrolysis of both model substrate, azo-casein and soybean meal protein (SMP). It was found that decreasing flux could improve the extent of hydrolysis and that a single-layer reactor can hydrolyze about 50% of the substrate to peptides with the molecular weight of 10 kDa or less. Hydrolysis of SMPs was demonstrated in a continuous five-layer BM reactor and both BMs showed excellent hydrolysis capacity. This study provides the groundwork for the development of high-efficiency BM for continuous and cost-effective protein hydrolysis for the production of value-added peptides.

11 Abstract Penicillin G acylases (PGAs) belong among enantioselective enzymes catalyzing a hydrolysis of stable amide bond in a broad spectrum of substrates, often having high application potential. PGAEc from Escherichia coli and PGAA from microorganism Achromobacter sp. CCM 4824 were used to catalyze enantioselective hydrolyses of seven selected N-phenylacetylated (N-PhAc) α/β-amino acid racemates. The PGAA showed higher stereoselectivity for three (S) enantiomers: N-PhAc-β-homoleucine, N-PhAc-α-tert- leucine and N-PhAc-β-leucine. We have constructed a homology model of PGAA that was used in molecular docking experiments with the same substrates. In-silico experiments reproduced the data from experimental enzymatic resolutions confirming validity of employed modeling protocol. We employed this protocol to evaluate enantiopreference of PGAA towards seven new substrates with application potential. For five of them, high enantioselectivity of PGAA was predicted for. PGAA was further studied in kinetically controlled syntheses of β-lactam antibiotics (SSBA). The PGAA was significantly more efficient at synthese of ampicillin and amoxicillin (higher S/H ratio and product accumulation) compared with PGAEc . Analogously to prediction of enantioselectivity of PGAA towards new substrates this protocol was applied...

Exploitation of well defined responsive polymer brushes for direct and controlled immobilization of metal/semiconductor nanoparticles on macroscopic surfaces has been demonstrated. The employed approach offers the possibility of the organization of a variety of inorganic nanoparticles by irreversible bonding and homogenous distribution on an underlying substrate. The immobilization process has been realized by chemical grafting of a variety of polymer brushes on a suitable substrate followed by the attachment of pre-/in-situ formed nanoparticles exploiting the chemical/physical interactions between surface functionalities of nanoparticles and polymer chain segments. A number of polymer brushes including poly (acrylic acid), polystyrene, poly (2-vinyl pyridine) and poly (N-isopropyl acrylamide) brushes have been prepared on silicon substrate by the “grafting to” approach. A variety of inorganic nanoparticles such as quantum dots (CdTe) noble metals (gold and silver) and magnetic (Fe3O4) were immobilized on macroscopic surfaces to impart them photo luminescent, catalytic or magnetic properties. In addition, responsiveness of grafted polymer brushes in terms of variation in thickness (due to changes in chain conformation) as a function of external stimuli such as solvent and pH allowed to use the resulting polymer brush-nanoparticles nanoassemblies in the fabrication of nanosensors. The design of fabricated nanosensors is based on the modulation in the interparticle distance of immobilized nanoparticles due to swelling/deswelling of underlined polymer brushes in response to some external trigger.

Skeletal muscle is a highly adaptable tissue that comprises approximately 40% of total body weight while accounting for up to 90% of whole-body oxygen consumption and energy expenditure during exercise. The loss of skeletal muscle protein and subsequent decrease in muscle mass (atrophy) that accompanies disuse results primarily from a decrease in intracellular protein synthesis combined with an increase in proteolytic activity. Interestingly, these processes of skeletal muscle atrophy are amplified by changes in mitochondrial capacity, with evidence suggesting that the maintenance of mitochondria during periods of disuse protects skeletal muscle against atrophy. Remarkably, rodents with denervated muscle are protected against muscle atrophy following whole-body heat stress. The mechanism of protection appears to be tied to the observed increases in heat shock protein (HSP) and PGC-1α, which accompany the heat stress. Without any published observations as to whether such heat-induced protection against muscle atrophy would translate to human muscle, the aim of this project was to determine the extent to which deep tissue heating (via pulsed shortwave diathermy) might provide protection against skeletal muscle atrophy.

Las etapas de transferencia electrónica o transferencia de carga involucradas en reacciones electroquímicas juegan un papel muy importante en un gran número de procesos biológicos y bioquímicos. Hoy en día, el interés de la comunidad científica se centra en explorar y entender exhaustivamente la naturaleza biológica y química de los fenómenos bioelectroquímicos que ocurren en los seres vivos, con el objeto de mimetizarlos en el laboratorio. Los procesos bioelectrocatalíticos presentan un amplio abanico de aplicaciones dirigidas al: (i) desarrollo de biorreactores electroquímicos para la mitigación de las emisiones de gases de efecto invernadero, la eliminación de contaminantes presentes en aguas residuales y urbanas, o la síntesis de productos con alto valor añadido para la industria, (ii) el desarrollo de biopilas y biobaterías, y (iii) el desarrollo de (bio)sensores electroquímicos con fines analíticos. Sin embargo, la implantación en el mercado de dispositivos basados en procesos biocatalíticos aún se enfrenta a varios desafíos, como son la robustez, la estabilidad a largo plazo, la reproducibilidad y la rentabilidad de producción en términos de materiales y fabricación de los dispositivos electroquímicos. La motivación de esta tesis doctoral es la de enfrentarse a algunos de los desafíos con los que se encuentra hoy en día la bioelectrocatálisis, para ello esta tesis doctoral se centra, principalmente en el estudio de nuevos materiales y mejora de rutas y estrategias bioelectrocatalíticas, con la finalidad de desarrollar dispositivos electroquímicos con aplicaciones analíticas y en la obtención de productos de valor añadido. En primer lugar esta tesis doctoral recoge el estudio y desarrollo de (bio)sensores electroquímicos para la determinación de lactato, L-cisteína, peróxido de hidrógeno y pH en medios biológicos complejos, y en segundo lugar estudia la bioelectrosíntesis de ácido fórmico a través de la reducción bioelectroquímica de dióxido de carbono.

Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical Technology Candidate Mgr. Jana Kubačková Supervisor Assoc. Prof. Jarmila Zbytovská, Mgr., Dr. rer. nat. Co-supervisor PharmDr. Ondřej Holas, PhD. Title of Doctoral Thesis Preparation of pharmaceutical formulations based on polymeric and lipid carriers Nanomedicine allows application of nanoscaled drug delivery carriers to achieve a therapy that can be tailored in terms of e.g. controlled release, site-specific delivery and protection of an active substance. From multiple nanoplatforms available for drug delivery, advantage was taken of biocompatible and biodegradable polymers and lipids to enable targeted intracellular delivery, delivery of a poorly water-soluble drug and delivery of a sensitive macromolecule. In the study with biodegradable polymeric nanomaterial we worked with experimental poly(lactic-co-glycolic acid) (PLGA) polymers. The formulations were optimised for targeting to phagocytic macrophages - of size up to 300 nm and negative surface charge. For this purpose, two linear and one branched PLGA were screened in combination with one of four surfactants in low concentrations (0.1-1%). These PLGA polymers were formulated into nanoparticles and loaded with a hydrophilic fluorescent dye Rhodamine B...