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@inproceedings{1678121,
author = {Filipi, T. and Mazura, P. and Dopitová, Radka and Janda, Lubomír and Damborský, Jiří and Kiran, N. S. and Brzobohaty, B.},
address = {BRNO},
booktitle = {MENDELNET 2010},
editor = {Skarpa, P.},
keywords = {Zm-p60.1; beta-glucosidase; maize; tZOG; cZOG; cytokinin; protein evolution; protein mutagenesis},
howpublished = {tištěná verze "print"},
language = {cze},
location = {BRNO},
isbn = {978-80-7375-453-2},
pages = {821-823},
publisher = {MENDEL UNIV BRNO, FAC AGRONOMY},
title = {DESIGNING RECOMBINANT MAIZE beta-GLUCOSIDASE Zm-p60.1: DEVELOPMENT OF NOVEL ENZYMES MODULATING CYTOKININ METABOLISM},
url = {https://mnet.mendelu.cz/mendelnet2010/articles/19_filipi_284.pdf},
year = {2010}
}
TY - JOUR
ID - 1678121
AU - Filipi, T. - Mazura, P. - Dopitová, Radka - Janda, Lubomír - Damborský, Jiří - Kiran, N. S. - Brzobohaty, B.
PY - 2010
TI - DESIGNING RECOMBINANT MAIZE beta-GLUCOSIDASE Zm-p60.1: DEVELOPMENT OF NOVEL ENZYMES MODULATING CYTOKININ METABOLISM
PB - MENDEL UNIV BRNO, FAC AGRONOMY
CY - BRNO
SN - 9788073754532
KW - Zm-p60.1
KW - beta-glucosidase
KW - maize
KW - tZOG
KW - cZOG
KW - cytokinin
KW - protein evolution
KW - protein mutagenesis
UR - https://mnet.mendelu.cz/mendelnet2010/articles/19_filipi_284.pdf
N2 - Maize beta-glucosidase Zm-p60.1 is one of many enzymes which are important for plant development. It liberates free zeatin from its transport and/or storage form zeatin-O-glucoside. Using an adapted site specific non-saturated random mutagenesis approach, it were prepared five multi-site mutants surrounding the active site (W373K/M376L, W373K/P372S/M376L, W373K/P372T/M376L, W373K/P372S and W373K/P372T) derived from the single mutant W373K to study the effect(s) of amino-acid changes on substrate specificity towards natural (trans-zeatin-O-beta-D-glucopyranoside and cis-zeatin-O-beta-D-glucopyranoside) and artificial (4-nitorophenyl-O-beta-D-glucopyranoside and 4-methylumbellyferyl O-beta-D-glucopyranoside) substrates. Kinetic and substrate specificity studies confirmed large differences among set of mutated enzymes. All enzymes surprisingly preferred cis-zeatin-O-beta-D-glucopyranoside over trans-zeatin-O-beta-D-glucopyranoside, whereas differences in hydrolytic efficiencies are considerable. Quantitative TLC confirmed the best cZOG/tZOG hydrolysis ratio toward cis-zeatin-O-beta-D-glucopyranoside in the triple mutant W373K/P372T/M376L. Moreover, it was also confirmed that only wild-type hydrolyzed trans-zeatin-N9-beta-D-glucopyranoside. No known plant beta-glucosidase hydrolyzes this substrate. Hydrolysis of trans-zeatin-N7-beta-D-glucopyranoside was not observed at all.
ER -
FILIPI, T., P. MAZURA, Radka DOPITOVÁ, Lubomír JANDA, Jiří DAMBORSKÝ, N. S. KIRAN and B. BRZOBOHATY. DESIGNING RECOMBINANT MAIZE beta-GLUCOSIDASE Zm-p60.1: DEVELOPMENT OF NOVEL ENZYMES MODULATING CYTOKININ METABOLISM. In Skarpa, P. \textit{MENDELNET 2010}. BRNO: MENDEL UNIV BRNO, FAC AGRONOMY, 2010, p.~821-823. ISBN~978-80-7375-453-2.
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