The doctoral thesis deals with the production and immobilization of ligninolytic enzymes produced by wood decay fungi. Part of the results was published in internationally reviewed journals with an impact factor and at conferences. The general point of view, this work has contributed to the development of knowledge in the area of immobilization, namely CLEA immobilization and the use of the immobilized enzyme to degrade organopollutans and toxic synthetic dyes commonly found as waste products of the textile industries. First part of this work examines the screening of suitable white rot fungal strains. In the study, 25 strains of white rot fungi were tested. Different copper concentrations were used as inducer of laccase activity and two different substrates (ABTS, syringaldazine). Trametes versicolor and Fomes fomentarius strains were chosen as the most promising strains for laccase production in liquid media and further testing demonstrated good degradation ability against synthetic dyes and contaminated wastewater. The second part is dedicated to the purification of enzymes (laccase) in two strains (Trametes versicolor, Fomes fomentarius). First, the laccase was precipitated with 75 wt% ammonium sulfate then the glutaraldehyde was used to form cross-linked CLEA enzyme aggregates. The third thematic area concerns the optimization of precipitation and preparation of CLEA immobilized laccase and application of free and immobilized CLEA laccase for the decolorization of synthetic dyes.

The diploma thesis deals with the issue of nanoscale Zero-Valent Iron (nZVI) application for the chemically assisted phytostabilization of zinc (Zn) in order to decrease mobility of this element in the environment. The interactions of nZVI and Zn have been investigated during the experiments using tomato plants (Lycopersicon esculentum L.), which were grown in a hydroponic solution. Furthermore, the effects of the interactions on the uptake of water, macronutrients and iron (Fe) have been assessed. The use of nZVI proved to be an effective solution for the stabilization of lower concentrations of Zn (100 micromol) in the root system or for purposes of the assisted phytoremediation. However, the impact of nZVI was not significant high concentrations of Zn (500 micromol). During the application of Zn at increased concentrations a decrease in water uptake has been observed, which can limit the macronutrient uptake. It is necessary to pay attention to further research and study the effects of the nanoparticles application and their interactions in the environment.

The main objective of the thesis was to evaluate the potential of selected Mn and Fe (nano)oxides for the stabilization of metals and metalloids in contaminated soils. The research was focused basically on three materials - commercial nanomaghemite (Fe III), nanomagnetite (Fe II,III) and a synthetic amorphous Mn oxide (AMO). The main aim of the work was to provide a complex view on the chosen stabilizing amendments regarding not just their direct influence on contaminants mobility and stabilization mechanisms, but also their stability and alterations in soil conditions together with influence on soil microorganisms and higher plants. Firstly, adsorption properties of the tested materials towards Cd, Cu, Pb and As were investigated. In this context, the most effective material showed to be the AMO reaching one to two orders of magnitude higher adsorption capacities than Fe III and Fe II,III under given experimental conditions. Interestingly, the rate of As(V) adsorption onto AMO was increasing with increasing pH as a result of high pHzpc of the AMO (8.1) and significant dissolution of this phase at lower pH values. As a next step, the influence of (nano)oxides on metal(loid)s mobility and other physico-chemical soil characteristics after application to contaminated soil was examined. Again, the AMO proved to be the most efficient in reducing mobile pools of Cd, Cu, Pb, Zn and As. On the other hand, Fe III and Fe II,III addition had generally less significant effects on contaminants mobility. AMO application further resulted in an increase of soil pH connected with AMO dissolution and unwanted decomposition of soil organic matter. When (nano)oxides alterations in soil conditions were observed, MnCO3 coatings were identified on AMO surface while no significant changes were recorded for Fe III and Fe II,III. As the MnCO3 formation was connected with increased AMO stability, AMO particles synthetically covered with MnCO3 coating (denoted as SM AMO) were prepared. Although the SM-AMO had a lower mass loss in soil than pure AMO, the stabilizing efficiency was almost the same for both materials. The differences in surface composition of both materials were decreasing with time as MnCO3 naturally precipitated on the AMO surface in soils while the SM AMO coating was gradually dissolving. When investigating the effect on soil microbiota, AMO efficiently promoted soil microbial activity while no significant changes were observed in the case of Fe III and Fe II,III. The AMO was also able to reduce the uptake of Cd, Pb and Zn by sunflower (Helianthus annuus L.), eliminate Zn phytotoxicity symptoms and increase biomass yield. On the other hand, toxic levels of Mn released from the AMO in an acidic soil were found in sunflower tissues. AMO application is thus recommended for contaminated neutral or slightly alkaline pH with a higher cation exchange capacity in order to avoid unwanted release of Mn. Finally, various types of AMO-biochar composite sorbents were recently prepared and field experiment focused on stabilization of Cd, Pb, Zn and As using studied materials is currently under preparation. The combined results from the thesis highlight the importance of a complex experimental approach dealing with all parts of the contaminated soil environment in order to obtain complete information about the efficiency and usefulness of any newly developed stabilizing amendment.

Ligninolytic systems found their application in the food industry, but they are also used for the degradation of several xenobiotic compounds and dyes. This characteristic is making them be a useful tools for bioremediation purposes, that is why the interest in ligninolytic enzymes is increasing. The main aim of practical part was preparation of immobilized enzyme lignin peroxidase by method CLEA from fungus Piptoporus betulinus, Trametes gibbosa and their cocultivation. Activities of free and immobilized enzymes from these fungus were compared and measured by UV/VIS spectrometer. Conditions of precipitation and immobilization were specified by method CLEA. pH optimum for proteins precipitation were for Piptoporus betulinus pH 7,0, for Trametes gibbosa pH 5,0 and for cocultivation pH 6,0. The best concentration of glutaraldehyd, as a crosslinking agent, was for Piptoporus betulinus and cocultivation 50mM and for Trametes gibbosa 10mM. By immobilization, the activity of all enzymes were successfully increased up by 20 %. After optimalization of CLEA enzymes preparation, other parameters such as pH optimum, stability and temperature optimum and stability were tested. All enzymes had their pH optimum in acidic to slightly acidic environment and temperature optimum was in range from 30 °C to 40 °C. Immobilized CLEA enzyme from cocultivation was the most stable for all the enzymes. Free and CLEA enzymes were tested on synthetic food dyes, while their biodegradable ability was examined. Decrease of dyes was measured on HPLC with DAD detector. The sorption of fungi play an important role in this experiment, because fungi created mycelium and partially sorbed the dye. From this point of view, higher decolorization of dye in free enzymes in comparison with CLEA was observed. The best ability of decolorization was shown by a free enzyme from a Trametes gibossa, which after 14days of cultivation decolored more than 90 % dye. In total, significant decolorization (more than 80 % during 14 days) was achieved for azorubin. Degradation of dyes by CLEA was not so effective, which could be possibly caused by cultivation of enzymes without their substrate (veratrylalcohol). Cocultivation technique is very promising method how to increase LiP activity and concurrently create enzyme with improved properties.

The ability of alumino-silicate systems to immobilize hazardous compounds has been investigated since 1990s. The aim of this work is to develope alumino-silicate system (matrix) based on industrial waste products with ability to immobilize hazardous compounds (heavy metals). This ability of the matrix was confirmed by leaching tests based on the law 294/2005 Sb. Concentration of heavy metals in leachates was determined by ICP-MS method. Alumino-silicate system prepared in this work consists of high-temperature fly ash and blast furnace slag activated by mixture of alkaline activators (hydroxide and water glass). Matrixes were characterized by suitable analytic methods (XRD, SEM, FT-IR, DTA-TGA-EGA). The same analytic methods were used to describe the mechanism of immobilization of selected elements (Ba, Cu, Pb) in prepared systems.

8 Abstract Epoxide hydrolases (EHs) demonstrating high degree of enantioselectivity or enantioconvergence are useful biocatalysts for the production of optically active epoxides and vicinal diols, which can serve as chiral building blocks for syntheses of biologically active drugs. EHs can play an important role also in degradations of xenobiotics. Genes encoding EHs Kau2 and Kau8 were expressed in E. coli host strains TOP10 and RE3. Enantioselectivities and regioselectivities of Kau2 and Kau8 in supernatants of desintegrated cells were determined for four substrates: tert-butylglycidyl ether, para-chlorostyrene oxide, para-nitrostyrene oxide, α-methylstyrene oxide. The highest values of enantioselectivity and regioselectivity were achieved with Kau2 and para-nitrostyrene oxide as a substrate. The Kau2 was chosen for further experiments on the basis of these results. Kau2 was overexpressed in the recombinant strain RE3(pSEKau2). We performed two batch cultures and one fed-batch culture in stirred bioreactor. The highest volumetric activity of 4500 U/l was obtained in the case of fed-batch culture. Two phase system consisting of polyethylenglycole 6000 and sodium citrate (pH 7.7) was used for Kau2 purification from the supernatant of desintegrated cells. Purification factor 2.6 +/- 0.3 was achieved and...

Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it...

This project assesses effects of four -week active rehabilitation combined with Vojta reflex stimulation on mobility of bed-bound seniors. Sixteen female patients were divided into two groups of eight members, i.e. tested group and control group. Both groups were put through training focused on self-support, maintaining or enhancement of range of movement, strengthening of weak muscle groups, stability training, practicing sitting up and lying down from sitting position, and walking in the high walking frame in the case of stronger patients. Besides that the tested group underwent Vojta reflex stimulation (global model of reflex turning), coordination and stability exercise and exercise with rehabilitation tools (elastic band, over ball, small ball, water bottle). In the beginning and in the end of the project the EMS (Elderly Mobility Scale), BI (Barthel Index), and MMSE (Mini-mental State Examination) scales were tested as well as ability to turn from back to side and stability in the sitting position. Statistical comparability of both groups was proved true in the beginning of the therapy by Mann-Whitney test. There was a noticeable tendency towards higher probability of enhancing measured scores in the tested group compared to the control group after the therapy. In case of the EMS this probability was...

In this work the influence of alkaline activator on immobilization of lead and copper in alkali-activated blast furnace slag was investigated. A total of five activators were used; sodium water glass, potassium water glass, sodium hydroxide, potassium hydroxide and sodium carbonate. The leaching test according to ČSN EN 12457-4 was used to evaluate the level of immobilization of heavy metals, the leached solutions were analyzed by ICP-OES. For a better understanding of immobilization, the selected samples were characterized by analytical methods (FTIR, XRD, SEM, XPS). It was found that the degree of immobilization Pb2+ and Cu2+ in AAS was very high, regardless of the type of alkaline activator used.

This diploma thesis deals with the preparation of solid hydrophilic sponges made of acetalized PVAl. The sponges were produced by foaming by gaseous reaction by-product. The experimental work also includes the study of active compound immobilization into the sponge (finely ground lignite as a heavy metal sorbent).