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Preparation and characterization of lignin modified by laccase enzyme
Hrušková, Lucie ; Obruča, Stanislav (referee) ; Kalina, Michal (advisor)
This master’s thesis focuses on the enzymatic modification of commercially available lignin using laccase enzyme and monitoring its impact on selected physical and chemical properties. Lignin is an attractive material for the production of renewable chemicals, materials, and energy, but its complex chemical structure poses a major obstacle to its effective utilization. Enzymatic modification appears to be a promising solution for its processing. Experiments were conducted under various conditions, such as pH, enzyme dosage, and reaction time. In the subsequent part of the experiment, the modified lignins were characterized and their results compared with those of the raw kraft lignin before modification. Molecular weight was determined using SEC-MALS, and the concentration of phenolic groups and antioxidant activity were measured. Structural changes were also monitored using FTIR. The results indicate that the enzymatic modification of lignin using laccase led to an increase in the molecular weight of the modified lignins, along with a decrease in the content of phenolic groups and a reduction in antioxidant activity. Based on the obtained results, it can be concluded that the action of the laccase enzyme resulted in lignin polymerization and the formation of new chemical bonds, leading to further aggregation.
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Texturní a barevné vlastnosti sýrů ošetřených extracelulárními enzymy
Jašková, Jana
This diploma thesis deals with textural and color properties of cheeses treated with an extracellular enzyme. The cheese samples were matured for three weeks. The penetration and compression method was used to determine the textural properties of the cheeses. The color of the cheeses was measured on a CM 3500d spectrophotometer. The lowest strength determined by the penetration method had the sample with laccase after production (0.55 ± 0.07 N) and the highest strength had the control sample, which was aged for one week (1.31 ± 0.14 N). The lowest strength determined by the compression method had the sample with laccase, which had matured for two weeks (3.11 ± 1.07 N) and the highest strength had the control sample of cheese matured for three weeks (12.28 ± 2.36 N). The values of L*(D65) varied for all samples in the range from 72.89 ± 0.31 to 87.76 ± 0.01, the values of a*(D65) from -1.95 ± 0.01 to 0.66 ± 0.05 and b*(D65) values were measured between 6.73 ± 0.35 and 20.93 ± 0.03 Laccase enzyme treatment had no significant effect on the firmness and color of the cheese samples.
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Vliv receptury a délky zrání na obsah polyaminů ve zrajících sýrech
Kolková, Andrea
The theoretical part of my thesis was focused on the characteristics of polyamines, their synthesis, degradation and effect in the organism. The practical part was involved on determining the content of polyamines (putrescine, spermine and spermidine) in ripening cheese without addition and with the addition of laccase. The high-performance liquid chromatography method was used for the analysis. Quantitatively, the most significant polyamine in all tested cheese variants was spermine (laccase in cheese grains 16.6 mg/kg > control 15.5 mg/kg > surface laccase 14.8 mg/kg; p < 0.05). Spermidine content was in the range (surface laccase 8.8 mg/kg > laccase in cheese grains 2.9 mg/kg > control 2.4 mg/kg; p < 0.05). Putrescine was detected only in low precipitation (laccase in cheese grains 2.1 mg/kg > control 1.3 mg/kg > laccase in surface 0.7 mg/kg; p < 0.05). During storage (X; weeks), the total PA content (Y; mg/kg) in the control cheese sample without the addition of laccase increased according to the equations: y = 256.93 + 207.38x (R² = 0.8955; p = 0.0021 ), the amount of PA in the sample of cheese with laccase in the cheese grains also increased linearly according to equation: y = 196.05 + 228.22x (R² = 0.8226; p = 0.0024). The total PA content in the cheese sample with laccase on the surface increased up to 4 weeks and then decreased according to the equation: y = 629.1 + 621.45x - 59.539x2 (R² = 0.8984; p = 0.0163). The effect of laccase on the degradation of amines described in the literature has not been proven. However, this is a pilot experiment that investigates the effect of laccase on the content of polyamines in ripening cheeses. For a better understanding of this problematics, will be needed more extensive research.
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Study of the effect of microbial degradation of lignin on its molecular weight
Fialová, Tereza ; Hudečková, Helena (referee) ; Kovalčík, Adriána (advisor)
This diploma thesis deals with the microbial degradation of lignin using wood-degrading fungi Fomitopsis pinicola and Lenzites betulina. In the first phase of the experimental part, media screening was carried out, the aim of which was to select the best media for fungal growth and production of ligninolytic enzymes. Based on the determination of activity, in the next step, the conditions for their production were optimized in selected media. As part of these experiments, the optimal concentration of lignin and glucose was selected for subsequent cultivations. Commercially available lignin was used as substrate. Based on the results of measured enzyme activities, Lenzites betulina was selected as the better producer. The most produced enzyme of this fungus was laccase, followed by manganese-dependent peroxidase and the least lignin peroxidase. Further experiments were performed only with L. betulina. In the next part of the experiment, lignin was characterized, and its properties compared before and after microbial degradation. Its molecular weight was determined by SEC-MALS and the change in structure by FTIR. The molecular weight of lignin decreased after cultivation and a change in its structure was also noted. The results show that the microbial degradation of lignin was successful.
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Study of the interaction between fungus Pleurotus ostreatus and bacterial cultures on the abiotic surfaces - morphological, biochemical and proteomic analysis
Kozická, Barbora ; Petráčková, Denisa (advisor) ; Konopásek, Ivo (referee)
Ligninolytic fungi are well known for their ability to degrade a wide range of xenobiotics contaminating the environment, including synthetic industrial dyes. In this work Pleurotus ostreatus was used for decolorization of a synthetic textile dye Remazol Brilliant Blue R (RBBR). To set up a model fungal "fixed-bed" bioreactor the fungus was immobilized on a polyurethane foam and artificially contaminated with a model bacterium Rhodococcus erythropolis. The development of bacterial contamination can be expected during a real application of fungal bio filters in wastewater treatment. The main aim of the work was to study interspecies interactions in the model bioreactors during the dye decolorization. Ligninolytic enzyme activities were followed in the bioreactor cultures as markers of fungal biodegradation ability. In contrast to the controls, no bacterial growth was observed in the P. ostreatus bioreactor culture liquid. The results showed that fungal laccase, pH of the culture liquid, and glucose consumption by the fungus had no effect on the bacterial growth. However, 4*105 - 1,3*106 CFU/ml of R. erythropolis was detected to be associated with the fungal solid support. The presence of these bacteria had no effect on the decolorization performance of the bioreactors. Dye decolorization efficiency...
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Studium produkce lakázy vybranými druhy hub
Ovchynnikova, Oleksandra
The importance of lignin-degrading fungi and their ligninolytic enzymes has been well appreciated globally, because of their potential use in various industries. This bachelor thesis is focused on study of potential production of extracellular ligninolytic enzymes by brown rot fungi. In the literary review selected strains of brown rot fungi and its most studied ligninolytic enzymes are characterized. The use of laccase in various industries especially food industry are summarized. Activity of ligninolytic enzymes produced by brown rot fungi Laetiporus sulphureus, Serpula lacrymans, Gloeophyllum sepiarium, Phaeolus schweinitzii and Laetiporus montanus during submerged cultivation for 24 days was evaluated. Laccase, lignin peroxidase and manganese dependent peroxidase activities were measured spectrophotometrically. For all enzymes, the highest activity was observed on the 12 day. Selected fungi produced the most lignin peroxidase. Laetiporus sulphureus and Gloeophyllum sepiarium showed the highest values of enzyme activities. It has been found that the enzymatic activity of fungi depends on the geographical location and the specific habitat. Enzyme activities of the same strain taken from different locations were different.
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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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Využití ligninolytických enzymů v potravinářství
Nadtochaeva, Polina
Ligninolytic enzymes are produced by wood-decaying fungi, and due to their high reactivity and low specificity, they have recently become important enzymes for biotechnological use. This thesis briefly reviews the most important ligninolytic enzymes and their reaction mechanisms and summarizes the ways of using these enzymes in the food industry. Great attention is paid to biosensors, which are used as modern analytical tools for the detection of phenolic compounds. In the experimental part, the own biosensor for the detection and determination of (+) catechin concentration was prepared. The measurement was carried out using a differential pulse voltammetry method using a carbon paste electrode prepared using the immobilized laccase from Trametes versicolor. Measurements of standard solutions have been performed to show that the biosensor can detect even relatively small amounts of catechin. Then the prepared biosensor was used for real samples of red wine and green teas. The content of catechin in four tea samples was measured. The concentrations were approximately the same and ranged from 19.57 to 24.91 mg·l-1. In the case of wine, it has been found that due to the complicated matrix, quantitative analysis is allowed, but requires appropriate mathematical processing of the results or pre-treatment of the samples.
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Využití ligninolytických enzymů v degradaci polutantů
Badinová, Ester
Ligninolytic fungi are known for their rich enzyme system. Their use in environ-mental treatment of pollutants has been studied for a long time, but the interaction of heavy metals with ligninolytic fungi and the possible effect that this interaction could have on the biodegradation process has not been fully described. In order to identify the potentially most suitable representatives of the white rot fungi for mycoremediation, a screening of 17 representatives of fungi isolated from Czech forests was carried out. To identify the appropriate mycorremedication candidates, the fungi were tested for various concentrations of the most frequent pollutants Cu + II, Cr + V, Pb + II and Zn + II for one week. Five representatives of Trametes hirsuta, Perennipora fraxinea, Ganoderma carnosum, Phellinus punctatus and Schizophyllum communae were selected, showing growth in the presence of all the above-mentioned metals. To verify the appropriate biodegradation potential, the enzymatic activities of laccase, lignin-peroxidase and manganese-dependent peroxidase produced by fungi in the contaminated soil were rec-orded spectrophotometrically, which were then compared to the control of non-heavy metal activities. Detected activity of laccase was 100 % higher in contaminated soil, 40 % higher in case of lignin peroxidase and 75 % higher in case of manganese-dependent peroxidase. The increase of activities could therefore be caused by the pres-ence of heavy metals that stimulated enzyme activities. Schizophyllum communae was suggested as the most suitable representative for mycorrection.
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Studium produkce a imobilizace ligninolytických enzymů u dřevokazných hub
Vršanská, Martina
The doctoral thesis deals with the production and immobilization of ligninolytic enzymes produced by wood decay fungi. Part of the results was published in internationally reviewed journals with an impact factor and at conferences. The general point of view, this work has contributed to the development of knowledge in the area of immobilization, namely CLEA immobilization and the use of the immobilized enzyme to degrade organopollutans and toxic synthetic dyes commonly found as waste products of the textile industries. First part of this work examines the screening of suitable white rot fungal strains. In the study, 25 strains of white rot fungi were tested. Different copper concentrations were used as inducer of laccase activity and two different substrates (ABTS, syringaldazine). Trametes versicolor and Fomes fomentarius strains were chosen as the most promising strains for laccase production in liquid media and further testing demonstrated good degradation ability against synthetic dyes and contaminated wastewater. The second part is dedicated to the purification of enzymes (laccase) in two strains (Trametes versicolor, Fomes fomentarius). First, the laccase was precipitated with 75 wt% ammonium sulfate then the glutaraldehyde was used to form cross-linked CLEA enzyme aggregates. The third thematic area concerns the optimization of precipitation and preparation of CLEA immobilized laccase and application of free and immobilized CLEA laccase for the decolorization of synthetic dyes.
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