prikaz prve stranice dokumenta Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases
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master's thesis
Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases
Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology, 2021. urn:nbn:hr:159:077009
Debanić, Fran
University of Zagreb
Faculty of Food Technology and Biotechnology
Department of Biochemical Engineering
Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology

Institutional repository: Repository of the Faculty of Food Technology and Biotechnology

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Debanić, F. (2021). Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases (Master's thesis). Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology. Retrieved from https://urn.nsk.hr/urn:nbn:hr:159:077009

Debanić, Fran. "Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2021. https://urn.nsk.hr/urn:nbn:hr:159:077009

Debanić, Fran. "Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases." Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, 2021. https://urn.nsk.hr/urn:nbn:hr:159:077009

Debanić, F. (2021). 'Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases', Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, accessed 09 December 2024, https://urn.nsk.hr/urn:nbn:hr:159:077009

Debanić F. Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases [Master's thesis]. Zagreb: University of Zagreb, Faculty of Food Technology and Biotechnology; 2021 [cited 2024 December 09] Available at: https://urn.nsk.hr/urn:nbn:hr:159:077009

F. Debanić, "Deciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases", Master's thesis, University of Zagreb, Faculty of Food Technology and Biotechnology, Zagreb, 2021. Available at: https://urn.nsk.hr/urn:nbn:hr:159:077009

Metadata
TitleDeciphering the enzymatic mechanisms of short-chain dehydrogenase/reductase epimerases and decarboxylases
Title (croatian)Istraživanje reakcijskih mehanizama kratkolančanih dehidrogenaza/reduktaza epimeraza i dekarboksilaza
AuthorFran Debanić
MentorAnita Slavica (mentor)
Committee memberMirela Ivančić Šantek (član povjerenstva)
GranterUniversity of Zagreb
Faculty of Food Technology and Biotechnology
(Department of Biochemical Engineering)
(Laboratory for Biochemical Engineering, Industrial Microbiology and Malting and Brewing Technology)
Zagreb
Defense date and country2021-07-23, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Abstract
UDP-GlcA methyl ester, a substrate analogue for short-chain dehydrogenase/reductase epimerases and decarboxylases - human UDP-Xylose synthase (UXS), UDP-Apiose/Xylose synthase (UAXS) and UDP-glucuronic acid 4-epimerase (UGAepi), was successfully synthesized and purified and thus enabled investigation of their enzymatic mechanisms. The three enzymes were expressed in E. coli and purified to homogeneity. Both decarboxylases (UXS and UAXS) converted UDP-GlcA methyl ester (0.71 U/mg and 0.03 U/mg, respectively) in a comparable manner to conversion of their natural substrate - UDP-glucuronic acid (UDP-GlcA, 1.6 U/mg and 0.15 U/mg, respectively). Newly established methanol assay proved that the two decarboxylases hydrolyze the ester before the decaborxylation reaction while UGAepi does not perform the hydrolysis. UGAepi epimerizes faster the substrate analogue (0.65 U/mg) than the natural substrate (0.53 U/mg). Additionally, gene encoding UAXS from Geminococcus roseus was successfully expressed in E. coli and resulting enzyme (GrUAXS) posses’ lower activity towards UDP-GlcA (2.1 mU/mg) than it's mutant (S120C; 12.3 mU/mg) supporting the importance of cysteine residue in the active site of the enzyme.
Abstract (croatian)
UDP-GlcA metil ester, analog supstrata za kratkolančane dehidrogenaze/redukataze epimeraze i dekarboksilaze - humanu UDP-ksiloza sintazu (UXS), UDP-apioza/ksiloza sintazu (UAXS) i UDP-glukuronska kiselina 4-epimerazu (UGAepi), uspješno je sintetiziran i pročišćen, čime je omogućeno istraživanje enzimskih mehanizama ovih katalizatora. Tri enzima su eksprimirana u stanicama E. coli i pročišćena. Obje dekarboksilaze (UXS and UAXS) su konvertirale UDP-GlcA metil ester (redom 0.71 U/mg i 0.03 U/mg) slično konverziji njihova prirodnog supstrata - UDP- glukuronske kiseline (UDP-GlcA, redom 1.6 U/mg i 0.15 U/mg). Pomoću novoustanovljene metode za određivanje metanola utvrđeno je da dvije dekarboksilaze hidroliziraju ester prije reakcije dekarboksilacije, dok UGAepi ne provodi hidrolizu estera. UGAepi brže epimerizira analog supstrata (0.65 U/mg) nego svoj prirodni supstrat (0.53 U/mg). Dodatno, gen koji kodira UAXS iz Geminococcus roseus je uspješno eksprimiran u E. coli i dobiveni enzim (GrUAXS) ima nižu aktivnost prema UDP-GlcA (2.1 mU/mg) nego njegov mutant (S120C, 12.3 mU/mg), što podupire važnu ulogu cisteina u aktivnom mjestu ovoga enzima
Keywords
Keywords (english)
Languageenglish
URN:NBNurn:nbn:hr:159:077009
Study programmeTitle: Bioprocess Engineering
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva (magistar/magistra inženjer/inženjerka bioprocesnog inženjerstva)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence Attribution 4.0 International (CC BY 4.0) icon
Created on2021-10-05 10:41:08