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Use of Chiral Separations for the Determination of Enzyme Enantioselectivity
Břicháč, Jiří ; Zima, Jiří (advisor) ; Tesařová, Eva (referee) ; Flieger, Miroslav (referee)
Conclusion Enantioselective enzymes pray important rore in metaboric pathways of chirar compounds. Enantioselective metabolism of lipid peroxidation product HNE was studied using measurement of enzyme kinetics. Difference in ability to detoxiý individua| HNE enantiomers by rat brain mitochondriar ALDHs was found. Based on kinetic parameters of purified recombinant enz),Ťnes' it was concluded, that rat ALDH5A enantioselective|y oxidized (R)-HNE' whereas rat ALDH2 was not enantioserective. Moreover, direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were deveroped and validated. The indirect method involved pre-corumn derivatization with a chiral amino agent ANPAD' and subsequent separation ofdiastereomers on a Spherisorb oDS2 column. The direct separation of HNEA enantiomers was performed using the porysacharide-based CSP, Chiralpak AD-RH. Enantioserective enzymes are useful biocatalyzators a'owing cheap preparation of optically pure chemicals. screening of epoxide hydrolases produced by various microorganisms was performed and enantioselective enzymes were found using chiral GC on cyclodextrin-based CSps. In summary, this thesis describes development and optimization of the analytical methods for determination of various chiral compounds implicated in enzyme enantioselectiviý. All...
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Use of Chiral Separations for the Determination of Enzyme Enantioselectivity
Břicháč, Jiří
Use of Chiral Separations for the Determination of Enzyme Enantioselectivity Jiří Břicháč Enantioselectivity is the ability of chiral environment to distinguish between two enantiomers. Enantioselective enzyme prefers one enantiomer as a substrate or preferentially forms one enantiomer over the other in an enzymatic reaction. Enantioselective enzymes are very useful biocatalyzators allowing cheap preparation of optically pure chemicals. In this work, we determined enantioselectivity of novel microbial epoxide hydrolases. Enantiopure epoxides prepared by an enzyme kinetic resolution of racemates can serve as valuable building blocks in an organic synthesis. In order to measure the enantioselectivity, we developed methods for analyses of various chiral epoxides using a chiral GC on cyclodextrine-based stationary phases. We showed, that epoxide hydrolase from Aspergillus niger M200 reacts with tert-butyl glycidyl ether in enantioselective manner.1 In the second part of this thesis, we elucidated the basis of enantioselective oxidation of trans-4-hydroxy-2-nonenal (HNE) by brain mitochondria.2 HNE is a cytotoxic product of lipid peroxidation involved in numerous diseases, including Alzheimer's disease. Here, we described enzyme kinetics of HNE enantiomers detoxification to trans-4- hydroxy-2-nonenoic acid...
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Use of Chiral Separations for the Determination of Enzyme Enantioselectivity
Břicháč, Jiří
Use of Chiral Separations for the Determination of Enzyme Enantioselectivity Jiří Břicháč Enantioselectivity is the ability of chiral environment to distinguish between two enantiomers. Enantioselective enzyme prefers one enantiomer as a substrate or preferentially forms one enantiomer over the other in an enzymatic reaction. Enantioselective enzymes are very useful biocatalyzators allowing cheap preparation of optically pure chemicals. In this work, we determined enantioselectivity of novel microbial epoxide hydrolases. Enantiopure epoxides prepared by an enzyme kinetic resolution of racemates can serve as valuable building blocks in an organic synthesis. In order to measure the enantioselectivity, we developed methods for analyses of various chiral epoxides using a chiral GC on cyclodextrine-based stationary phases. We showed, that epoxide hydrolase from Aspergillus niger M200 reacts with tert-butyl glycidyl ether in enantioselective manner.1 In the second part of this thesis, we elucidated the basis of enantioselective oxidation of trans-4-hydroxy-2-nonenal (HNE) by brain mitochondria.2 HNE is a cytotoxic product of lipid peroxidation involved in numerous diseases, including Alzheimer's disease. Here, we described enzyme kinetics of HNE enantiomers detoxification to trans-4- hydroxy-2-nonenoic acid...
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Use of Chiral Separations for the Determination of Enzyme Enantioselectivity
Břicháč, Jiří ; Zima, Jiří (advisor) ; Tesařová, Eva (referee) ; Flieger, Miroslav (referee)
Conclusion Enantioselective enzymes pray important rore in metaboric pathways of chirar compounds. Enantioselective metabolism of lipid peroxidation product HNE was studied using measurement of enzyme kinetics. Difference in ability to detoxiý individua| HNE enantiomers by rat brain mitochondriar ALDHs was found. Based on kinetic parameters of purified recombinant enz),Ťnes' it was concluded, that rat ALDH5A enantioselective|y oxidized (R)-HNE' whereas rat ALDH2 was not enantioserective. Moreover, direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were deveroped and validated. The indirect method involved pre-corumn derivatization with a chiral amino agent ANPAD' and subsequent separation ofdiastereomers on a Spherisorb oDS2 column. The direct separation of HNEA enantiomers was performed using the porysacharide-based CSP, Chiralpak AD-RH. Enantioserective enzymes are useful biocatalyzators a'owing cheap preparation of optically pure chemicals. screening of epoxide hydrolases produced by various microorganisms was performed and enantioselective enzymes were found using chiral GC on cyclodextrin-based CSps. In summary, this thesis describes development and optimization of the analytical methods for determination of various chiral compounds implicated in enzyme enantioselectiviý. All...
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