|
|
Microbial lipases and their application
Pavlačková, Jana ; Flodrová, Dana (referee) ; Omelková, Jiřina (advisor)
This diploma thesis is focused on the study of the preparation for fat separators and wastewater pipes that contains the microorganisms with lipolytic activity. Theoretical part of this thesis describes lipases, microorganisms producing this enzymes and usage of lipases. In this part possibilities of identification of microorganisms are presented too. The practical part is concerned with the study of commercial preparation Sany Duo Spezial with proven presence of microorganisms with lipolytic activity. These microorganisms were identified by means of the PCR method. This method identified mictoorganisms like genus Bacillus sp. Next characterization of the preparation was focused on the determination of COD and the investigation of the influence of various conditions of culture medium on the lipases production and their activity. The effect of temperature, ions and pH was studied. Lipolytic activity was determine spectrophotometricaly with usage of p-nitrophenyllaurate whitch dissociates to yellow product p-nitrophenol.
|
|
|
Properties and production of microbial lipases
Martinková, Patrícia ; Reichstädter, Marek (referee) ; Omelková, Jiřina (advisor)
Bachelor´s thesis is focused on testing the culture media for growing various strains of yeasts, producing lipolytic enzymes and to study the influence of culture media composition on the production of lipolytic enzymes. Theoretical part of this thesis states the characteristics of lipolytic enzymes, their properties, sources, conditions for their production and the possibilities of their application. Together 8 strains of yeasts were used in the experimental part of the thesis, namely 2 strains of Yarrowia lipolytica, 2 strains of Kluyveromyces lactis, Cryptococcus saitoi, Candida intermedia, Candida oleophila and Debaryomyces hansenii, which were cultivated on 4 media with different compounds. During the cultivation the growth of biomass was monitored and growth curves were formed based on these results. Selected strains of yeasts were tested during the process of cultivation for lipolytic activity of two types of enzymes – enzymes bonded to the cellular structure and extracellular enzymes, which were measured by spectrophotometer. Production of lipolytic enzymes varied depending on applied culture medium.
|
|
|
Study of preparations for fat separators
Gojkovic, Živan ; Ing.Dana Flodrová, Ph.D. (referee) ; Omelková, Jiřina (advisor)
Diploma thesis was concentrated at testing of a single commercial product designed as a lipid residue removal agent, applicable in grease trap vessel. Concept was, that product is to be tested for possible content of viable microbial culture with ability to utilize various kinds of vegetable oils and animal fats, by producing functional lipase. Four types of vegetable oils were used, and two fats of animal origin: olive oil, palm oil, rapeseed oil, sunflower oil, lard and beef fat. Determined fat characteristics were: point of the saponification, the acid value, the ester value and the peroxide value. Submerged cultivation was performed on specially composed medium in which the oil or fat of choice, was mixed later. Lipolytic activity and biomass growth measurement was performed using spectrophotometry, lipid degradation ability was measured using titrimetry. Based on results it can be stated that tested microorganisms, originated from product, and its lipase has ability to successfully utilize all of used fats and oils and thus, theoretically, remove its content from grease trap.
|
|
|
Optimization of the Production of Lipases by Bacillus subtilis
Slavíčková, Radka ; Omelková, Jiřina (referee) ; Hermanová, Soňa (advisor)
In this thesis, optimization of production of lipolytic enzymes by submerzed cultivation of Bacillus subtilis (BS) was studied. Production of lipolytic enzymes was tested in three nutrient media, which differed mainly in main sources of carbon, respectively of nitrogen. The first medium contained mainly extract from calf brain and beef heart (BHIB), the second medium contained peptone and yeast extract (NB) and the third one contained peptone and yeast extract with the addition of 2% (w/v) glucose (NBG). The highest lipolytic activity (0.0784 Uml-1) was measured in NBG medium. Maximum of lipolytic activity was observed before the end of the exponential phase of BS growth in all the media. Temperature optimum in NBG medium was determined from 30 to 50 °C, pH optimum in the range of 5 to 11 and subsequently the temperature stability of lipolytic enzymes produced by the BS was estimated. The activity value was determined spectrometrically using p-nitrophenyllaurate as a substrate. Produced lipolytic enzymes showed maximum activity at 37 °C in the alkaline pH of 8.0. Measurement of temperature stability showed that lipolytic enzymes are relatively thermostable enzymes retaining 100 % of the activity even after 1 hour of cultivation at 30 - 50 °C. The presence of 1% (w/v) olive oil in medium NBG caused a decrease in lipolytic activity by 65 % as well as in pH from 6.5 to 5.4 after 14 days of cultivation. After substitution of glucose by fructose in medium NBG, lipolytic activity showed comparable values during the first week of cultivation. On the other hand, the decrease of lipolytic activity by 29 % in the medium with fructose was observed after 14 days of cultivation. A procedure for the identification of lipolytic enzymes of BS by peptide massfingerprinting was developed to understand the potential of synthetic polyester - poly(e-caprolactone) as a lipase inductor. Degradation study of commercial polyester poly(e-caprolactone) was carried out by submerged cultivation of Bacillus subtilis in NBG medium at initial pH 7.0 and 30 °C for 14 days. PCL (Mn = 10,000, Mw = 14 000) was studied in the form of films (1.0 x 1.0 cm), which were prepared by melt-pressing, rapid cooling of the melt to 4 °C and evaporation of the solvent from 2 % dichlormethane solution. The evaluation of the films shown occurrence of weight loss (7.8 - 17.0 wt.%) together with the formation of numerous holes and cracks in the sample surface in relation to the method of the films preparation. Lipolytic activity values increased by 9 - 17 % in the degradation media compared to control samples. Densitometric monitoring showed also higher increase in cell mass in the degradation medium compared with control samples. Based on the results obtained, the degradation process induced by BS could be suggested.
|
|
|
The study of extracellular enzymes produced by different species of yeast
Vršanská, Martina ; Ing.Jana Molnárová, Ph.D. (referee) ; Voběrková, Stanislava (advisor)
The thesis deals with the study of the different yeast strains from the point of view of extracellular lipolytic enzyme production. First part of this work consisting of appropriate yeasts was developed within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. From ten given strains three yeasts such as Pseudozyma fusiformata, Meyerozyma guilliermondii, Yarrowia lipolytica were chosen, these strains showed the highest lipolytic activity and cell growth on basal medium with Tween 80. These yeasts were used for optimization of cultivation conditions and characterization of lipolytic enzymes. The yeasts were cultivated on media with different carbon sources, which appeared to be a most suitable medium the basal medium with Tween. Tween acted as and inducer of lipase production. The substrate specificity was determined using three p-nitrophenylester substrates with varying sizes of the fatty acid site chains. The results showed that tested lipases are probably triacylglycerol-acyl-hydrolases which has the highest activity towards in the water insoluble substrates with medium long chains. The pH optimum and temperature optimum were measured. The results showed that the tested lipases had the highest activity in neutral and mild acid region around 30°C. By measuring of thermal stability has been demonstrated that extracellular lipases are relatively thermostable enzymes. Afterwards the storage stability was measured for 5 weeks when supernatant was kept in fridge at 4°C and in freezing box at -20°C. The results showed that in both cases tested lipases exhibited high storage stability which allows to store the samples without loss of activity for a longer time. Finally, the results of lipolytic and proteolytic activity, cell growth and pH of the medium of yeast Y. lipolytica were compared between the batch cultivation in L-tubes with the continual cultivation in the bioreactor. The highest lipases production was achieved in bioreactor due to the setting conditions of the continual proces to regulate the production and enzymatic stability.
|
|
|
Degradation of fats in wastes
Artýszková, Jana ; Flodrová, Dana (referee) ; Omelková, Jiřina (advisor)
Submitted master’s thesis is focused on the study of possibilities of lipids degradation in particular wastewaters originating in food industry or restaurants. The effort is given to the employment of lipolytic activity presenting microorganisms. In the literature review, wastewater treatment with aim on the sludge management and fat separators are described, as a way how to pre-treat these wastewaters. In this part the enzymes lipases of microbial origin are researched from point of view of their production conditions and possible applications. The experimental part is dedicated to the research of optimization of cultivation conditions for lipases production employing selected microorganisms (Bacillus subtilis, Geobacillus thermodenitrificans, G. thermocatenulatus and mixed bacterial culture Thermus and Bacillus) and a commercial formulation (Sany Duo Spezial). Lipases production and growth of microorganisms are determined spectrofotometrically on various concentrations of lipids. Moreover, employing the solid nutrition medium, the effect of detergents onto the Bacillus subtilis culture was assessed, since detergents are generally abundant in this particular wastewaters. As a conclusion, vide supra mentioned microorganisms were characterized according to their abilities to degrade triacylglycerols.
|
|
|
Properties and production of microbial lipases
Martinková, Patrícia ; Reichstädter, Marek (referee) ; Omelková, Jiřina (advisor)
Bachelor´s thesis is focused on testing the culture media for growing various strains of yeasts, producing lipolytic enzymes and to study the influence of culture media composition on the production of lipolytic enzymes. Theoretical part of this thesis states the characteristics of lipolytic enzymes, their properties, sources, conditions for their production and the possibilities of their application. Together 8 strains of yeasts were used in the experimental part of the thesis, namely 2 strains of Yarrowia lipolytica, 2 strains of Kluyveromyces lactis, Cryptococcus saitoi, Candida intermedia, Candida oleophila and Debaryomyces hansenii, which were cultivated on 4 media with different compounds. During the cultivation the growth of biomass was monitored and growth curves were formed based on these results. Selected strains of yeasts were tested during the process of cultivation for lipolytic activity of two types of enzymes – enzymes bonded to the cellular structure and extracellular enzymes, which were measured by spectrophotometer. Production of lipolytic enzymes varied depending on applied culture medium.
|
|
|
The study of extracellular enzymes produced by different species of yeast
Vršanská, Martina ; Ing.Jana Molnárová, Ph.D. (referee) ; Voběrková, Stanislava (advisor)
The thesis deals with the study of the different yeast strains from the point of view of extracellular lipolytic enzyme production. First part of this work consisting of appropriate yeasts was developed within study interships in Slovak Academy of Sciences, department of Glycomics in Bratislava. From ten given strains three yeasts such as Pseudozyma fusiformata, Meyerozyma guilliermondii, Yarrowia lipolytica were chosen, these strains showed the highest lipolytic activity and cell growth on basal medium with Tween 80. These yeasts were used for optimization of cultivation conditions and characterization of lipolytic enzymes. The yeasts were cultivated on media with different carbon sources, which appeared to be a most suitable medium the basal medium with Tween. Tween acted as and inducer of lipase production. The substrate specificity was determined using three p-nitrophenylester substrates with varying sizes of the fatty acid site chains. The results showed that tested lipases are probably triacylglycerol-acyl-hydrolases which has the highest activity towards in the water insoluble substrates with medium long chains. The pH optimum and temperature optimum were measured. The results showed that the tested lipases had the highest activity in neutral and mild acid region around 30°C. By measuring of thermal stability has been demonstrated that extracellular lipases are relatively thermostable enzymes. Afterwards the storage stability was measured for 5 weeks when supernatant was kept in fridge at 4°C and in freezing box at -20°C. The results showed that in both cases tested lipases exhibited high storage stability which allows to store the samples without loss of activity for a longer time. Finally, the results of lipolytic and proteolytic activity, cell growth and pH of the medium of yeast Y. lipolytica were compared between the batch cultivation in L-tubes with the continual cultivation in the bioreactor. The highest lipases production was achieved in bioreactor due to the setting conditions of the continual proces to regulate the production and enzymatic stability.
|
|
|
Optimization of the Production of Lipases by Bacillus subtilis
Slavíčková, Radka ; Omelková, Jiřina (referee) ; Hermanová, Soňa (advisor)
In this thesis, optimization of production of lipolytic enzymes by submerzed cultivation of Bacillus subtilis (BS) was studied. Production of lipolytic enzymes was tested in three nutrient media, which differed mainly in main sources of carbon, respectively of nitrogen. The first medium contained mainly extract from calf brain and beef heart (BHIB), the second medium contained peptone and yeast extract (NB) and the third one contained peptone and yeast extract with the addition of 2% (w/v) glucose (NBG). The highest lipolytic activity (0.0784 Uml-1) was measured in NBG medium. Maximum of lipolytic activity was observed before the end of the exponential phase of BS growth in all the media. Temperature optimum in NBG medium was determined from 30 to 50 °C, pH optimum in the range of 5 to 11 and subsequently the temperature stability of lipolytic enzymes produced by the BS was estimated. The activity value was determined spectrometrically using p-nitrophenyllaurate as a substrate. Produced lipolytic enzymes showed maximum activity at 37 °C in the alkaline pH of 8.0. Measurement of temperature stability showed that lipolytic enzymes are relatively thermostable enzymes retaining 100 % of the activity even after 1 hour of cultivation at 30 - 50 °C. The presence of 1% (w/v) olive oil in medium NBG caused a decrease in lipolytic activity by 65 % as well as in pH from 6.5 to 5.4 after 14 days of cultivation. After substitution of glucose by fructose in medium NBG, lipolytic activity showed comparable values during the first week of cultivation. On the other hand, the decrease of lipolytic activity by 29 % in the medium with fructose was observed after 14 days of cultivation. A procedure for the identification of lipolytic enzymes of BS by peptide massfingerprinting was developed to understand the potential of synthetic polyester - poly(e-caprolactone) as a lipase inductor. Degradation study of commercial polyester poly(e-caprolactone) was carried out by submerged cultivation of Bacillus subtilis in NBG medium at initial pH 7.0 and 30 °C for 14 days. PCL (Mn = 10,000, Mw = 14 000) was studied in the form of films (1.0 x 1.0 cm), which were prepared by melt-pressing, rapid cooling of the melt to 4 °C and evaporation of the solvent from 2 % dichlormethane solution. The evaluation of the films shown occurrence of weight loss (7.8 - 17.0 wt.%) together with the formation of numerous holes and cracks in the sample surface in relation to the method of the films preparation. Lipolytic activity values increased by 9 - 17 % in the degradation media compared to control samples. Densitometric monitoring showed also higher increase in cell mass in the degradation medium compared with control samples. Based on the results obtained, the degradation process induced by BS could be suggested.
|
|
|
Degradation of fats in wastes
Artýszková, Jana ; Flodrová, Dana (referee) ; Omelková, Jiřina (advisor)
Submitted master’s thesis is focused on the study of possibilities of lipids degradation in particular wastewaters originating in food industry or restaurants. The effort is given to the employment of lipolytic activity presenting microorganisms. In the literature review, wastewater treatment with aim on the sludge management and fat separators are described, as a way how to pre-treat these wastewaters. In this part the enzymes lipases of microbial origin are researched from point of view of their production conditions and possible applications. The experimental part is dedicated to the research of optimization of cultivation conditions for lipases production employing selected microorganisms (Bacillus subtilis, Geobacillus thermodenitrificans, G. thermocatenulatus and mixed bacterial culture Thermus and Bacillus) and a commercial formulation (Sany Duo Spezial). Lipases production and growth of microorganisms are determined spectrofotometrically on various concentrations of lipids. Moreover, employing the solid nutrition medium, the effect of detergents onto the Bacillus subtilis culture was assessed, since detergents are generally abundant in this particular wastewaters. As a conclusion, vide supra mentioned microorganisms were characterized according to their abilities to degrade triacylglycerols.
|
guest ::
