The Czech Environmental Inspectorate

This thesis aims to introduce the Czech Environmental Inspectorate as a specialized body of the state administration with a state-wide authority which is subordinated to the Ministry of the Environment. The objective of the Inspectorate is particularly to exercise state supervision connected with enforcement of corrective actions and sanctions. The Czech Environmental Inspection was established in 1991 with its original competence in a field of forest protection, as of today it has competences with respect to all environmental components. The merit of this thesis is an analysis of statutory regulations regarding the protection of the environment according to which the Inspectorate exercises its competences and authority. The result of this analysis is definition of the competences of the Inspectorate in individual fields of environment protection. In the thesis I also focus on problematic provisions of particular legal regulations and in final chapter I address also a possibility of reorganisation of the Inspectorate in the future

Biocatalyzed Reactions towards Functional Food Components 4-Alkylcatechols and Their Analogues

Catechols are antioxidants and radical scavengers with a broad medical potential. 4-Methylcatechol (1b) and 4-ethylcatechol (2b) (occurring in some traditional fermented and smoked foods) activate the cell defense against oxidative stress. We examined the biocatalyzed reactions towards 4-n-alkylcatechols with different side chains length, which is a factor important for the biological activities of catechols. 4-n-Alkylcatechols with methyl through heptyl side chains (1b–7b) were obtained in one pot by (i) oxidation of phenols 1a–7a with tyrosinase from Agaricus bisporus followed by (ii) reduction of ortho-quinones (intermediates) with L-ascorbic acid sodium salt. The conversions decreased with increasing side chain length. The preparative reactions were carried out with substrates 1a–5a. The isolated yields of the purified products decreased from 59% in 2b to 10% in 5b in correlation with logP of the substrates. Homology modeling indicated that the affinities of two tyrosinase isoforms (PPO3 and PPO4) to the substrates with side chains longer than C2 decreased with increasing side chain length. This was probably due to steric limitations and to missing interactions of the extended side chains in the active sites. We envisage using the model to predict further substrates of tyrosinase and testing the products, catechols, for radical-scavenging and biological activities

Plant Nitrilase Homologues in Fungi: Phylogenetic and Functional Analysis with Focus on Nitrilases in Trametes versicolor and Agaricus bisporus

Fungi contain many plant-nitrilase (NLase) homologues according to database searches. In this study, enzymes NitTv1 from Trametes versicolor and NitAb from Agaricus bisporus were purified and characterized as the representatives of this type of fungal NLase. Both enzymes were slightly more similar to NIT4 type than to NIT1/NIT2/NIT3 type of plant NLases in terms of their amino acid sequences. Expression of the synthetic genes in Escherichia coli Origami B (DE3) was induced with 0.02 mM isopropyl β-D-1-thiogalactopyranoside at 20 °C. Purification of NitTv1 and NitAb by cobalt affinity chromatography gave ca. 6.6 mg and 9.6 mg of protein per 100 mL of culture medium, respectively. Their activities were determined with 25 mM of nitriles in 50 mM Tris/HCl buffer, pH 8.0, at 30 °C. NitTv1 and NitAb transformed β-cyano-L-alanine (β-CA) with the highest specific activities (ca. 132 and 40 U mg−1, respectively) similar to plant NLase NIT4. β-CA was transformed into Asn and Asp as in NIT4 but at lower Asn:Asp ratios. The fungal NLases also exhibited significant activities for (aryl)aliphatic nitriles such as 3-phenylpropionitrile, cinnamonitrile and fumaronitrile (substrates of NLase NIT1). NitTv1 was more stable than NitAb (at pH 5–9 vs. pH 5–7). These NLases may participate in plant–fungus interactions by detoxifying plant nitriles and/or producing plant hormones. Their homology models elucidated the molecular interactions with various nitriles in their active sites