|
| |
|
| |
|
| |
|
| |
|
| |
|
| |
|
|
Study of function and molecular architecture of fungal nitrilases applicable in biocatalysis
Veselá, Alicja Barbara ; Martínková, Ludmila (advisor) ; Macek, Tomáš (referee) ; Teisinger, Jan (referee)
Nitrilases are enzymes which catalyze the hydrolysis of a nitrile into the corresponding carboxylic acid and ammonia. These enzymes are potentially applicable in biocatalysis and bioremediation because of their advantages over the conventional (chemical) methods of nitrile hydrolysis (lower demand for energy, safety, simplicity, high yields, selectivity). In this work, genome mining was used to search for the sequences of hypothetical nitrilases from filamentous fungi. The amino acid sequences of previously characterized fungal nitrilases were used as the templates. Then the new synthetic genes together with other genes from our nitrilase library were expressed in E. coli and the substrate specificities of the enzymes thus produced were compared. Significant attention was focused on the relationships between the sequence of the enzyme and its substrate specificity. The arylacetonitrilases from Arthroderma benhamiae (NitAb) and Nectria haematococca (NitNh) were purified and characterized. Their substrate specificities, kinetic parameters, pH and temperature profiles and subunit and holoenzyme size were assessed. NitAb and NitNh together with other recombinant fungal nitrilases were employed in the hydrolysis of high concentrations of (R,S)-mandelonitrile in a batch or fed-batch mode. Nitrilase from...
|
|
|
Structure and biocatalytic properties of nitrilases from filamentous fungi
Křenková, Alena ; Martínková, Ludmila (advisor) ; Macek, Tomáš (referee) ; Klempier, Norbert (referee)
Filamentous fungi are alongside bacteria a rich source of nitrilases that can be used for biotechnological purposes. There is a large number of sequences of putative nitrilases in databases, but few of these enzymes have been expressed, purified and characterized. According to the sequences in GenBank database, synthetic genes of putative nitrilases from Aspergillus niger CBS 513.88 and Neurospora crassa OR74A were designed, expressed in Escherichia coli and the nitrilases produced were purified and characterized. The effect of chaperone-assisted nitrilase expression was also examined using a set of commercial chaperone plasmids. The structural properties of nitrilases were examined using various methods, such as homology modelling, sedimentation velocity analysis or electron microscopy. Recombinant nitrilase production was compared to their production by native producers - filamentous fungi. The cultivation of native producers was rather difficult and nitrilase production was very low, therefore, the advantage of using prokaryotic expression system was highlighted. Nitrilases with amino acids close to the catalytic cysteine replaced were prepared in order to study the effects of certain amino acids on the activity, chemoselectivity and enantioselectivity of nitrilases. The catalytic properties of...
|
|
| |
|
|
Nitrile hydrolysing enzymes: Fungal nitrilases
Vejvoda, Vojtěch ; Martínková, Ludmila (advisor) ; Wimmer, Zdeněk (referee) ; Vosáhlová, Simona (referee)
Charles University in Prague Faculty of Science Department of Biochemistry A thesis submitted for the degree of Doctor of Philosophy Nitrile-hydrolysing enzymes: Fungal nitrilases Mgr. Vojtěch Vejvoda Supervisor: Ing. Ludmila Martínková, CSc. Laboratory of Biotransformation, Institute of Microbiology, v.v.i. Academy of Sciences of the Czech Republic Consultant: Prof. RNDr. Karel Bezouška, DrSc. Faculty of Science, Department of Biochemistry, , Charles University in Prague Prague 2010 INTRODUCTION Nitriles are widespread in the environment - not only as the natural products of plants but also as the secondary metabolites of microorganisms or higher organisms. Synthetic nitriles and inorganic cyanides occur as pollutants from human activities (industrial wastes, herbicides, etc.). Both natural and man-made nitriles are poisonous to many organisms, the natural ones serving as a protection against enemies (e.g. herbivores). Their toxic activity is used in some herbicides. However, all of these compounds may exert negative effects on other beings living in the particular environment and create selection pressures, probably leading to the evolution of enzymes catalyzing the detoxification of nitriles, or, even better, utilizing them as the source of energy and nutrients. Nitrile-metabolizing enzymes are...
|
guest ::
RSS feed.