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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.
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