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New possibilities of nitrilases in biocatalysis and bioremediation
Veselá, Alicja Barbara ; Bezouška, Karel (advisor) ; Weignerová, Lenka (referee)
Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...
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Function of sigma factors in strains of genus Rhodococcus
Nový, David ; Štěpánek, Václav (advisor) ; Sudzinová, Petra (referee)
Thanks to their large genome sizes, rhodococci share considerable biotechnological potential, however, there is still little known about the regulation of gene expression during the initiation of transcription process. The knowledge of this process might help to optimize biotechnological application. A large number of sigma factors are involved in the transcriptional regulation of rhodococci and this number often positively correlates with their genome sizes. Sigma factors can be potentially divided into two groups: the first, conserved factors that may in some variants occur in many different species among bacteria, and the second, specific sigma factors, that have been evolutionarily selected in individual strains due to environmental and lifestyle conditions. All sigma factors present in rhodococci belong to the σ70 family and can also be divided into four groups according to the presence of conserved structural domains (groups 1 to 4). A special type of sigma factors with extracytoplasmic function (ECF, group 4) are factors carrying specific domains at their C-terminus, namely NTF- 2-like or TPR, whose functions are still not fully understood. Very little information about the physiological functions of individual sigma factors in rhodococci has been published but it can be supplemented by...
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Responses of Rhodococcus strains to heavy metal stress
Volf, Jan ; Štěpánek, Václav (advisor) ; Martínková, Ludmila (referee)
Increasing heavy metal pollution can be dealt with not only through physical and chemical decontamination processes but also utilizing microbial bioremediation, a much more environmentally friendly way. Members of genus Rhodococcus are suitable candidates for effective bioremediations of heavy metals due to their considerable adaptability and extreme resistance to different stress conditions, including those related to presence of heavy metals or their toxic compounds. Individual rhodococcal strains are more resistant to heavy metals than most of other microorganisms and they are capable of biotransformating them to less toxic forms or at least of accumulating effectively or adsorbing them on produced polysaccharides and specific surface active substances. Their bioremediation potential is very high even though, so far, only molecular mechanisms of rhodococcal resistances to toxic arsenic compounds have been studied more in detail.
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Stress responses in Rhodococcus strains
Křenková, Lucie ; Pátek, Miroslav (advisor) ; Cajthaml, Tomáš (referee)
Throughout the evolution, bacteria developed ingenious mechanisms which help them survive and grow in harsh conditions, where extreme temperatures, pH, changing water and osmotic activity occurs. Rhodococcus sp. cope with these conditions so efficiently they are able to grow in the presence of toxic compounds. Large genome of Rhodococcus sp. consists of a great amount of genes involved in a broad spectre of catabolic pathways. To use these skills in biotechnology, it is necessary to know stress response mechanisms well. In response to the changing environment Rhodococcus sp. use transcriptional regulators, two-component system and RNA polymerase sigma factors to tune the expression profiles of the genes. The upregulation of chaperons, chaperonins and proteases is the main pattern followed in most of the stress responses. Vast majority of stress responses is complex and groups of genes to react on them interfere too. Thus, it is very difficult to interpret the data of these studies. However, the great importance of understanding these mechanisms is unquestionable. Key words: stress response, bacteria, Rhodococcus, biotechnology, degradation, toxic compounds, sigma factor
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Detekce fytopatogenů jahodníku
MICHÁLKOVÁ, Barbora
Strawberries were tested for presence of selected phytopathogens (Agrobacterium, Rhodococcus, phytoplasmas, SMYEV, SMoV, SPV1, SCV and SCRh1) by the PCR. At the same time was researched the transmission of mentioned phytopathogens by grafting. The DNA of the strawberries with positive results was sequenced.
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Expression of genes for the conversion of nitriles and amides in Rhodococcus erythropolis
Kracík, Martin ; Pátek, Miroslav (advisor) ; Mikušová, Gabriela (referee)
The strain Rhodococcus erythropolis A4 is a source of enzymes nitrilhydratase and amidase, that catalyse conversion of nitriles and amides. These enzymes are used in industrial biotransformation and bioremediation. Since it was difficult to carry out genetic manipulations aimed at increasing the production of these enzymes in the strain A4, the corresponding genes (ami and nha1 + nha2) of a related strain R. erythropolis CCM2595, in which both plasmid and chromosome manipulations can be routinely performed, were identified and analyzed in this diploma theses. The ami and nha1 + nha2 genes from the strain R. erythropolis CCM2595 were isolated and sequenced together with the flanking regions (5.5 kb in total). The organization of these genes and the expected regulatory genes was described in the strain CCM2595 and mechanisms of regulation of expression of these genes were studied. For the analysis of transcription of amidase and nitrilhydratase genes from both strains of R. erythropolis, the promoter-probe vector pEPR1 replicating in Escherichia coli and R. erythropolis was used. Transcriptional fusion of Pami promoters of the strains A4 and CCM2595 and the reporter gfp gene were constructed. The activity of the Pami promoter was measured by means of fluorescence of gfp gene product (green fluorescent...
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New possibilities of nitrilases in biocatalysis and bioremediation
Veselá, Alicja Barbara ; Bezouška, Karel (advisor) ; Weignerová, Lenka (referee)
Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...
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