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Title:ČIŠČENJE IN KARAKTERIZACIJA ENCIMA TREHALOZE FOSFORILAZE IZ GLIVE SCHIZOPHYLLUM COMMUNE
Authors:ID Heržič, Katja (Author)
ID Potočnik, Uroš (Mentor) More about this mentor... New window
Files:.pdf UNI_Herzic_Katja_2010.pdf (3,99 MB)
MD5: 4213F95E91681915215A7249BDE35179
PID: 20.500.12556/dkum/7d5cb456-0cc8-4e76-adda-aea9c4cb239d
 
Language:Slovenian
Work type:Undergraduate thesis
Organization:FKKT - Faculty of Chemistry and Chemical Engineering
Abstract: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.
Keywords:glikoziltransferaze, Schizophyllum commune, trehaloza fosforilaza, α, α-trehaloza, čiščenje, kinetični mehanizem, kinetični parametri
Place of publishing:Maribor
Publisher:[K. Heržič]
Year of publishing:2010
PID:20.500.12556/DKUM-16081 New window
UDC:66.098:577.15(043.2)
COBISS.SI-ID:14725654 New window
NUK URN:URN:SI:UM:DK:2R7PPQFU
Publication date in DKUM:30.09.2010
Views:3474
Downloads:221
Metadata:XML DC-XML DC-RDF
Categories:KTFMB - FKKT
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Secondary language

Language:English
Title:PURIFICATION AND CHARACTERIZATION OF SCHIZOPHYLLUM COMMUNE TREHALOSE PHOSPHORYLASE
Abstract: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.
Keywords:glycosyltransferases, Schizophyllum commune, trehalose phosphorylase, α, α-trehalose, purification, kinetic mechanism, kinetic parameters


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