|
|
Using of modern molecular methods for isolation and identification of ligninolytic enzymes
Řiháček, Martin
Ligninolytic enzymes are able to decay the structure of lignin. This effect can be useful in the industry (food industry, textile industry, farming, etc) because it can replace regular chemical processes. The white-rot fungus Phanerochaete chrysosporium is known for the production of these enzymes. This bachelor thesis deals with the identification and characterization of the behavior of enzymes such as lignin peroxidase, laccase and manganese peroxidase under different concentrations of copper sulfate (0, 0.1, 0.5 and 1 mM). For isolation of DNA and RNA, the fungi were grown in potato dextrose broth (PDB) during 5 days. Common PCR, reverse transcription PCR and quantitative real-time PCR were chosen for this experimental part. The PCR products were purified and sent to sequencing to confirm how their different isoforms develop under stress conditions of different concentrations of copper sulfate. Moreover, the enzymatic activity assay for the enzymes was done also under different copper sulfate environment for the experimental part. 1 mM of copper sulfate concentration influenced the transcription of the enzymatic genes resulting in the production of their isoforms. It was also observed at the level of the gene expression, with a higher expression of these 3 genes; laccase, manganese peroxidase and lignin peroxidase, compared with the control samples. On the other hand, the best conditions for carrying out their enzymatic activities were observed at 0.5 mM concentration of CuSO4 for lignin peroxidase and manganese peroxidase and 1 mM in the case of laccase. After the molecular characterization, we can conclude that production of the enzymes of Phanerochaete chrysosporium are affected by high copper concentrations.
|
|
|
Application of ligninolytic fungi on solid substrates for degradation of endocrine disrupters
Slavíková - Amemori, Anna ; Cajthaml, Tomáš (advisor) ; Gabriel, Jiří (referee)
Today a lot of attention is focused on compounds called endocrine disrupters (EDs) among substances released to environment by humans. They are a group of substances which can disturb function of hormonal system of organisms including humans. Their poor removal at wastewater treatment plants (WwTP) were shown at various studies, thus they can reach the environment in water. A prospective way for the degradation of EDs at WwTP can be their removal by ligninolytic fungi. They are able to degrade lots of lignin-like aromatic substances because of their highly nonspecific enzymes. In this work growth and enzyme production capability of four ligninolytic fungal strains were monitored on three solid substrates (straw pellets, poplar sawdust mixed with straw pellets, oak sawdust with straw pellets), which may be suitable substrates for fungal growth in bioreactors for wastewater treatment. Ability of these enzymes to degrade EDs were tested in in-vitro degradation experiment. Trametes versicolor was found as best degrading strain with 20 μg/ml of bisphenol A, 17 α- ethynylestradiol and nonylphenol degraded below a quantification limit within 24 hours. Fungal strains degraded EDs well on all of the three substrates but wood sawdust seemed to be a better substrate for fungal growth because straw pellets...
|
guest ::
