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Functional analysis of insect fatty acyl-coenzyme A reductases and desaturases
Tupec, Michal ; Pichová, Iva (advisor) ; Šulc, Miroslav (referee) ; Doležel, David (referee)
Fatty acid-derived lipids are an important class of biomolecules. In addition to their primary role in cellular architecture, energy storage and signaling, they function in many other ways, e.g. as isolating or waterproof coatings, defense compounds and pheromones. Multiple enzymes mod- ify intermediates originating from fatty acid biosynthetic pathway, including desaturases (FADs) which synthesize unsaturated fatty acids, and reductases (FARs) which synthesize fatty alcohols. Functionally highly diversified fatty acyl-modifying enzymes from insects, one of the most abun- dant animals in the world, present a significant source for modern biotechnology of cell factories. This thesis summarizes available information on the FADs and FARs and describes the results which we have obtained while studying them. In bumblebees, we identified several FAR transcripts which are abundantly expressed in male pheromone-producing tissue. We then functionally characterized the corresponding enzymes in yeasts, and estimated their participation in the biosynthesis of fatty alcohols observed in the mark- ing pheromone-producing tissue. The studied enzymes reduce broad range of substrates, from short fatty acyls (e.g. C14) to very long ones (e.g. C26), from saturated fatty acyls to polyunsatu- rated ones. We also found...
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Antioxidant properties of medicinal plants
Tupec, Michal ; Hýsková, Veronika (advisor) ; Poljaková, Jitka (referee)
During the evolution plants have developed rather efficient mechanisms for the re- moval of harmful compounds, which include e. g. the reactive oxygen species (ROS). These mechanisms are mainly based on the antioxidant effects of ascorbic acid, gluta- thione in intimate cooperation with superoxide dismutase, catalase and various perox- idases - the reduced coenzymes, NADH and NADPH, play a major role in the ROS elimination, too. However, in recent times, the attention has turned from these key compounds of primary metabolism to secondary metabolites. Interestingly, the phenolic com- pounds together with some terpenes and alkaloids exhibit antioxidant effects, too, and apparently they play an irreplaceable role in the life of a plant - besides their antiox- idant action they are also important in the fight against pathogens and herbivores, in attracting the pollinators or in the moderation of UV light effects. Natural products are also studied in their relation to man - a broad variety of effects is known including the antioxidant, anti-inflammatory and anticancer effects for the most part. In this work, extracts comprising mainly of low-molecular-weight compounds were obtained from 32 plants with either hot water, 80 %w aqueous methanol or just by plain juicing. The extracts were then examined by three...
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Chemo-enzymatic synthesis of antiviral prodrugs
Tupec, Michal ; Stiborová, Marie (advisor) ; Indra, Radek (referee)
Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it...
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Chemo-enzymatic synthesis of antiviral prodrugs
Tupec, Michal ; Stiborová, Marie (advisor) ; Indra, Radek (referee)
Lipases have been widely applied in the manufacture of food products and in some areas of the industry, nowadays they are used in synthetic organic chemistry catalyzing the hydrolytic/esterification reactions under very mild conditions in the field of protecting groups or enantiomer resolution. In this study, the commercial lipase from bacterium Pseudomonas fluorescens was immobilized using the sol-gel process into organosilicate materials with propyl, octyl or phenyl substituents. The highest hydrolytic activity was found in the enzyme on the octyl-derived carrier. The immobilized enzymes differ in their hydrolytic activities on 4-nitrophenyl esters of various lengths. Subsequent experiments revealed quite good pH stability of the enzymes in a buffer (incubations in pH 3 through pH 11), as well as good temperature stability in isooctane (incubations at up to 100 řC). The majority of organic solvents seem to have no substantial effect on the lipase activity. The biocatalytic properties were studied on a model compound from the group of the acyclic nucleoside analogues - 9-(2',3'-dihydroxypropyl)adenine (DHPA). It was found for example that the best acyl donors are vinyl esters, that the lipase shows a preference towards longer vinyl esters, that the reaction proceeds faster in non-polar solvents or that it...
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Antioxidant properties of medicinal plants
Tupec, Michal ; Hýsková, Veronika (advisor) ; Poljaková, Jitka (referee)
During the evolution plants have developed rather efficient mechanisms for the re- moval of harmful compounds, which include e. g. the reactive oxygen species (ROS). These mechanisms are mainly based on the antioxidant effects of ascorbic acid, gluta- thione in intimate cooperation with superoxide dismutase, catalase and various perox- idases - the reduced coenzymes, NADH and NADPH, play a major role in the ROS elimination, too. However, in recent times, the attention has turned from these key compounds of primary metabolism to secondary metabolites. Interestingly, the phenolic com- pounds together with some terpenes and alkaloids exhibit antioxidant effects, too, and apparently they play an irreplaceable role in the life of a plant - besides their antiox- idant action they are also important in the fight against pathogens and herbivores, in attracting the pollinators or in the moderation of UV light effects. Natural products are also studied in their relation to man - a broad variety of effects is known including the antioxidant, anti-inflammatory and anticancer effects for the most part. In this work, extracts comprising mainly of low-molecular-weight compounds were obtained from 32 plants with either hot water, 80 %w aqueous methanol or just by plain juicing. The extracts were then examined by three...
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