20.500.12556/DKUM-16081 ČIŠČENJE IN KARAKTERIZACIJA ENCIMA TREHALOZE FOSFORILAZE IZ GLIVE SCHIZOPHYLLUM COMMUNE PURIFICATION AND CHARACTERIZATION OF SCHIZOPHYLLUM COMMUNE TREHALOSE PHOSPHORYLASE Encim trehaloza fosforilaza je šele pred kratkim bila predstavljena kot široko razširjen encim v naravi. Encim je klasificiran kot član družine encimov glikoziltransferaze 4. Encim katalizira reverzibilno fosforilazo α,α-trehalozo in fosfat (Pi) in proizvaja D-glukozo in α-D-glukozo-1-fosfat kot produkte. Trehaloza je nereducirajoč disaharid, ki se pojavlja v velikem številu organizmov, od bakterij, gliv, različnih prokariontov in rastlin do brezvretenčarjev. Encim trehaloza igra tako pomembno vlogo kot hranitelj karbohidratov, kot tudi pri preprečevanju stresa, predvsem kot blažilec med vročinskim stresom telesa in dehidracijo. Glikoziltransferaze so velikokrat zmotno prikazane kot neaktivne, saj na pogled privzemajo zgolj eno izmed dveh oblik. Veliko število nukleotidnih donorjev, velika izbira akceptorjev in zelo veliko število produktov pa kaže na enega izmed najvariabilnejših ogrodij v naravi. Skoraj neskončno število različnih produktov glikoziltransferaz, nam omogoča predvideti funkcije skoraj tisočih jasno definiranih struktur encimov. Zato nam bo le zadostno število struktur in karakterizacij omogočilo nabrati dovolj znanja o predvidenih funkcijah mnogih genov, ki kodirajo glikoziltransferaze. The enzyme Trehalose phosphorylase (ScTPase; EC 2.4.1.231) is a member of family 4 glycosyltrasferases (GT-4) found in fungi Schizophyllum commune. ScTPase catalyses the reversible phosphorolysis of α,α-trehalose and phosphate into α-D-glucose 1-phosphate and D-glucose. The α,α-trehalose is most widely distributed disaccharide which could be often found in vegetative cells and spores of fungi, where it has a protective function on unstable compounds against deactivation. Enzymes responsible for the breakdown of α,α-trehalose are trehalase and trehalose phosphorylase. For understanding and examinating the structure-function relationships of ScTPase, the gene encoding the enzyme was cut out from an old pQE 30 vector and inserted into a new pASK IBA 7+ vector with BamHI and PstI restriction endonucleases. The gene was than sequenced and expressed in E. coli JM109 cells.The enzyme was purified, using different purification techniques, including Strep-Tag purification column, anion exchange DEAE column, gel filtration column and NAP column. With the purified enzyme, kinetic studies were performed, determining important kinetic parameters, such as KM values and kcat values. Those kinetic studies were determined for phosphorolysis as well as for the synthesis direction of the reaction. We found that the recombinant ScTPase has a catalytic center activity of 4,53 s-1for the phosphorolysis and 3,05 s-1 for the synthesis of α,α-trehalose at 30 °C. Catalytic center activities (kcat) and Michaelis constants (KM) for the substrates were very similar for both phosphorylase forms. Our results suggest ScTPase operates by a sequential rather than coincidental kinetic mechanism. glikoziltransferaze Schizophyllum commune trehaloza fosforilaza α α-trehaloza čiščenje kinetični mehanizem kinetični parametri glycosyltransferases Schizophyllum commune trehalose phosphorylase α α-trehalose purification kinetic mechanism kinetic parameters true true false [K. Heržič] Slovenski jezik Angleški jezik Diplomsko delo 2010-09-29 22:52:35 2010-09-30 14:20:36 2022-04-13 07:51:11 2023-12-20 03:16:15 2010 0 Maribor 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 66.098:577.15(043.2) 14725654 URN:SI:UM:DK:2R7PPQFU UNI_Herzic_Katja_2010.pdf UNI_Herzic_Katja_2010.pdf 1 4213F95E91681915215A7249BDE35179 ec8432b4b350ae6a73a44ae11e0dd46e0d6a7a35b3430e0108b54453c11d0d3b 8f48ffa1-7c04-11eb-bb7a-00155d0001ca 20.500.12556/dkum/7d5cb456-0cc8-4e76-adda-aea9c4cb239d https://dk.um.si/Dokument.php?lang=slv&id=18032 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0