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Production of lignin-degrading enzymes by Phanerochaete chrysosporium
Péčiová, Bianka ; Hudečková, Helena (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis deals with the study of the production of ligninolytic enzymes produced by the mold P. chrysosporium and subsequently their immobilization. The theoretical part describes the selected production microorganism, principles of action of ligninolytic enzymes (lignin-peroxidase, manganese-dependent peroxidase and laccase) and methods of their immobilization. In the experimental part, optimal conditions of the production of ligninolytic enzymes by P. chrysosporium were monitored as a composition of culture medium with different glucose content and with using two different inducers: lignin and guaiacol. During the 14-day cultivation cycles, the enzyme activity of lignin-peroxidase, manganese-dependent peroxidase and laccase was monitored on each day of cultivation. Based on the optimized enzyme production procedure, the enzymes were subsequently immobilized by various methods: by method of forming enzyme aggregates (CLEA), adsorption on bacterial cellulose (BC) and a combination of both (CLEA + BC). The CLEA method proved to be the most effective, but there was problem with isolation of enzyme aggregates. Therefore, immobilization on bacterial cellulose associated with the enzymes aggregates was chosen as the most advantageous. With this method, the operational stability of the immobilized enzymes was monitored in 7 consecutive reaction cycles. Subsequently, CLEA and CLEA with BC were used to modify the model phenolic substance, where the alkali lignin was selected. For both sets of immobilizers was confirmed the effect of the enzymes on the lignin structure by measuring the molecular weight (Mw) and polydispersity index (PI).
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Imobilizácia lignolytických enzýmov
Schlosserová, Nikola
Ligninolytic systems found their application in the food industry, but they are also used for the degradation of several xenobiotic compounds and dyes. This characteristic is making them be a useful tools for bioremediation purposes, that is why the interest in ligninolytic enzymes is increasing. The main aim of practical part was preparation of immobilized enzyme lignin peroxidase by method CLEA from fungus Piptoporus betulinus, Trametes gibbosa and their cocultivation. Activities of free and immobilized enzymes from these fungus were compared and measured by UV/VIS spectrometer. Conditions of precipitation and immobilization were specified by method CLEA. pH optimum for proteins precipitation were for Piptoporus betulinus pH 7,0, for Trametes gibbosa pH 5,0 and for cocultivation pH 6,0. The best concentration of glutaraldehyd, as a crosslinking agent, was for Piptoporus betulinus and cocultivation 50mM and for Trametes gibbosa 10mM. By immobilization, the activity of all enzymes were successfully increased up by 20 %. After optimalization of CLEA enzymes preparation, other parameters such as pH optimum, stability and temperature optimum and stability were tested. All enzymes had their pH optimum in acidic to slightly acidic environment and temperature optimum was in range from 30 °C to 40 °C. Immobilized CLEA enzyme from cocultivation was the most stable for all the enzymes. Free and CLEA enzymes were tested on synthetic food dyes, while their biodegradable ability was examined. Decrease of dyes was measured on HPLC with DAD detector. The sorption of fungi play an important role in this experiment, because fungi created mycelium and partially sorbed the dye. From this point of view, higher decolorization of dye in free enzymes in comparison with CLEA was observed. The best ability of decolorization was shown by a free enzyme from a Trametes gibossa, which after 14days of cultivation decolored more than 90 % dye. In total, significant decolorization (more than 80 % during 14 days) was achieved for azorubin. Degradation of dyes by CLEA was not so effective, which could be possibly caused by cultivation of enzymes without their substrate (veratrylalcohol). Cocultivation technique is very promising method how to increase LiP activity and concurrently create enzyme with improved properties.
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