Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases
Samo za registrovane korisnike
2013
Autori
Blazic, Marija BKovacević, Gordana
Prodanović, Olivera
Ostafe, Raluca
Gavrovic-Jankulović, Marija D
Fischer, Rainer
Prodanović, Radivoje
Članak u časopisu (Objavljena verzija)
Metapodaci
Prikaz svih podataka o dokumentuApstrakt
Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, r...espectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.
Ključne reči:
Saccharomyces cerevisiae / High throughput screening / Glucose oxidase / Directed evolution / ChimeraIzvor:
Protein Expression and Purification, 2013, 89, 2, 175-180Izdavač:
- Academic Press Inc Elsevier Science, San Diego
Finansiranje / projekti:
- Ministry of Education and Science, Republic of Serbia
- Alergeni, antitela, enzimi i mali fiziološki značajni molekuli: dizajn, struktura, funkcija i značaj (RS-MESTD-Basic Research (BR or ON)-172049)
- Ispitivanja odnosa struktura-funkcija u ćelijskom zidu biljaka i izmene strukture zida enzimskim inženjeringom (RS-MESTD-Basic Research (BR or ON)-173017)
DOI: 10.1016/j.pep.2013.03.014
ISSN: 1046-5928
PubMed: 23562736
WoS: 000319373300009
Scopus: 2-s2.0-84876377170
Institucija/grupa
Institut za multidisciplinarna istraživanjaTY - JOUR AU - Blazic, Marija B AU - Kovacević, Gordana AU - Prodanović, Olivera AU - Ostafe, Raluca AU - Gavrovic-Jankulović, Marija D AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2013 UR - http://rimsi.imsi.bg.ac.rs/handle/123456789/705 AB - Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid. PB - Academic Press Inc Elsevier Science, San Diego T2 - Protein Expression and Purification T1 - Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases EP - 180 IS - 2 SP - 175 VL - 89 DO - 10.1016/j.pep.2013.03.014 ER -
@article{ author = "Blazic, Marija B and Kovacević, Gordana and Prodanović, Olivera and Ostafe, Raluca and Gavrovic-Jankulović, Marija D and Fischer, Rainer and Prodanović, Radivoje", year = "2013", abstract = "Glucose oxidase (GOx) catalyzes the oxidation of glucose to form gluconic acid and hydrogen peroxide, a reaction with important applications in food preservation, the manufacture of cosmetics and pharmaceuticals, and the development of glucose monitoring devices and biofuel cells. We expressed Aspergillus niger wild type GOx and the B11 mutant, which has twice the activity of the wild type enzyme at pH 5.5, as C-terminal fusions with the Saccharomyces cerevisiae Aga2 protein, allowing the fusion proteins to be displayed on the surface of yeast EBY100 cells. After expression, we extracted the proteins from the yeast cell wall and purified them by ion-exchange chromatography and ultrafiltration. This produced a broad 100-140 kDa band by denaturing SDS-PAGE and a high-molecular-weight band by native PAGE corresponding to the activity band revealed by zymography. The wild type and B11 fusion proteins had k(cat) values of 33.3 and 61.3 s(-1) and K-m values for glucose of 33.4 and 27.9 mM, respectively. The pH optimum for both enzymes was 5.0. The kinetic properties of the fusion proteins displayed the same ratio as their native counterparts, confirming that yeast surface display is suitable for the high-throughput directed evolution of GOx using flow cytometry for selection. Aga2-GOx fusion proteins in the yeast cell wall could also be used as immobilized catalysts for the production of gluconic acid.", publisher = "Academic Press Inc Elsevier Science, San Diego", journal = "Protein Expression and Purification", title = "Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases", pages = "180-175", number = "2", volume = "89", doi = "10.1016/j.pep.2013.03.014" }
Blazic, M. B., Kovacević, G., Prodanović, O., Ostafe, R., Gavrovic-Jankulović, M. D., Fischer, R.,& Prodanović, R.. (2013). Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. in Protein Expression and Purification Academic Press Inc Elsevier Science, San Diego., 89(2), 175-180. https://doi.org/10.1016/j.pep.2013.03.014
Blazic MB, Kovacević G, Prodanović O, Ostafe R, Gavrovic-Jankulović MD, Fischer R, Prodanović R. Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases. in Protein Expression and Purification. 2013;89(2):175-180. doi:10.1016/j.pep.2013.03.014 .
Blazic, Marija B, Kovacević, Gordana, Prodanović, Olivera, Ostafe, Raluca, Gavrovic-Jankulović, Marija D, Fischer, Rainer, Prodanović, Radivoje, "Yeast surface display for the expression, purification and characterization of wild-type and B11 mutant glucose oxidases" in Protein Expression and Purification, 89, no. 2 (2013):175-180, https://doi.org/10.1016/j.pep.2013.03.014 . .