|
|
A method for identification of foreign amylases in honey
Erban, Tomáš ; Shcherbachenko, Elena ; Talacko, Pavel ; Harant, Karel
Honey is a unique natural product. Honey has been used as a sweet and delicious foodstuff since ancient times. However, it is also valued for its multifaceted currative properties. Unfortunately, honey is one of the most adulterated foods. Nothing may be added to or modified from the honey. Honey also needs to be handled with care. Honey quality can negatively be affected by the way of processing such as heating and storage. Despite great progress in analytical methods, it is not possible to prove all adulterated honeys. Some methods of adulteration are quite sophisticated. Therefore, it is necessary find new approaches and methods for identification of honey adulteration. To be sold, honey must comply with internationally valid standards, which are also valid at national levels with possible minor modifications. One of the important parameters for honey is the level of diastase or amylase enzyme activity, which is a recognized indicator of the freshness and quality of honey. Lower diastase activity below the established level may indicate old honey, but it may also be the result of careless handling of honey. Last but not least, diastase activity may be reduced due to adulteration of honey such as its dilution with sugar substitutes. It is possible that amylase activity can be artificially adjusted by the addition of enzymes. Honey adulterated by the artificially added amylase meets the legislative requirements for placing honey on the market, but this violates the rules laid down by law. The methods used so far have not made it possible to prove this way of honey adulteration. Therefore, this methodology focuses on the identification of foreign amylases that may occur in honey. The methodology enables to identify practically any foreign amylase in honey by bottom-up shotgun proteomic approach. Based on the obtained results with specific peptides can be further used for the development of a targeted method for the identification of foreign amylases.
Fulltext:  PDF
|
|
|
The development of new tyrosin binding cross-linkers for structural characterization of proteins
Karpíšek, Michael ; Kukačka, Zdeněk (advisor) ; Talacko, Pavel (referee)
Proteins are cornerstones of all living organisms. A lot of energy is invested in studying structures of proteins, because their function is determined by their structure. One of the techniques used to access the structure of proteins is chemical cross-linking in combination with mass spectrometry. In spite of their number, most of the cross-linkers bind few aminoacids such as lysine, cysteine or glutamic and aspartic acid. In our work we tried to develop tyrosin-binding cross-linkers. Using top down and bottom up approaches we studied the influence of N-hydroxysuccinimide esters and imidazole on the reactivity of the cross-linker in different pH on model proteins. According to the acquired data there is a difference in overal reactivity and specifity between the used cross-linkers. The cross-linkers containing imidazole modified tyrosine more although the difference was small and binding of tyrosine was not selective. [IN CZECH]
|
|
|
Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Entlicher, Gustav (referee)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
|
|
|
Reprodukční a biotechnické metody využitelné v chovu dojeného skotu
Talácko, Pavel
The bachelor thesis describes the most frequent and best known biotechnical methods used in breeding of dairy cattle. It collects insemination methods useful in cattle breeding and familiarizations with their practical use. Because the rectal method is the most commonly used in practice, the most emphasis is on it. Next is an embryotransfer, which is described in the work, gets the embryo, the evaluation, the conservation until the transfer. This methods is closely related to superovulation and synchronization. Both of these methods are also described here. The other methods mentioned are in-vitro fertilization and sexed sperm, which is relatively often used today. All these methods show theirs advantages but also theirs disadvantages. In the next part of the thesis goes towards detecting the estrus. First of all, it describes the estrus and their symptoms. It is important monitor of estrus, which is of course also mention in the work. We can also find the tools that helps detect the estrus. Another part of this work is related reproduction indicators. The calving interval, servise period, insemination interval, insemination index, percent of pregnancy after 1. insemination are described here. Conclusion presents current values of these indicators in the Czech Republic and their comparison with abroad.
|
|
|
Cathepsin L from the hard tick Ixodes ricinus: analysis of proteolytic activity and its regulation
Talacko, Pavel ; Konvalinka, Jan (advisor) ; Ryšlavá, Helena (referee)
The hard tick Ixodes ricinus is an important blood-feeding parasite that transmits tick- borne diseases, such as tick-borne encephalitis and Lyme disease. Ticks employ a battery of proteolytic enzymes, including cathepsins, to digest their bloodmeal. These proteins are potential targets for the development of anti-tick vaccines. This work is focused on cathepsin L from I. ricinus (IrCL), namely its isoenzymes IrCL1 and IrCL3. IrCL1 was expressed in Pichia pastoris and chromatographically purified. Its substrate specifity was determined by the cleavage of (a) peptide fluorogenic substrates and (b) protein substrates analyzed by mass spectrometry. The proteolytic activity of IrCL1 was modulated by its interaction with glycosaminoglycans, which affected the pH optimum value. Futhermore, a proteolytically active mutant of IrCL1 with reduced number of N-glycosylation sites was prepared; this form will be used for crystallization experiments. IrCL3 was expressed in Escherichia coli, refolded and activated to its active form. The proteolytic activity of IrCL3 is in many ascpects similar to that of IrCL1, including substrate specifity, acidic pH optimum and activity modulation by glycosaminoglycans. Key words: cysteine proteases, cathepsin L, hard tick I. ricinus, substrate specifity, proteolytic activity...
|
|
|
Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
|
guest ::
RSS feed.