TY  - JOUR
AU  - Blazic, Marija
AU  - Balaž, Ana Marija
AU  - Prodanović, Olivera
AU  - Popović, Nikolina
AU  - Ostafe, Raluca
AU  - Fischer, Rainer
AU  - Prodanović, Radivoje
PY  - 2019
UR  - http://rimsi.imsi.bg.ac.rs/handle/123456789/1240
AB  - Featured Application Developed fluorescent assay and expression system can be used for obtaining improved cellobiose dehydrogenase whole cell biocatalysts for lactobionic acid production and building of biosensors and biofuel cells. Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10 mM lactose and 0.08 mM cellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.
PB  - MDPI, Basel
T2  - Applied Sciences-Basel
T1  - Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay
IS  - 7
VL  - 9
DO  - 10.3390/app9071413
ER  - 

@article{
author = "Blazic, Marija and Balaž, Ana Marija and Prodanović, Olivera and Popović, Nikolina and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje",
year = "2019",
abstract = "Featured Application Developed fluorescent assay and expression system can be used for obtaining improved cellobiose dehydrogenase whole cell biocatalysts for lactobionic acid production and building of biosensors and biofuel cells. Cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium can be used in lactobionic acid production, biosensor for lactose, biofuel cells, lignocellulose degradation, and wound-healing applications. To make it a better biocatalyst, CDH with higher activity in an immobilized form is desirable. For this purpose, CDH was expressed for the first time on the surface of S. cerevisiae EBY100 cells in an active form as a triple mutant tmCDH (D20N, A64T, V592M) and evolved further for higher activity using resazurin-based fluorescent assay. In order to decrease blank reaction of resazurin with yeast cells and to have linear correlation between enzyme activity on the cell surface and fluorescence signal, the assay was optimized with respect to resazurin concentration (0.1 mM), substrate concentration (10 mM lactose and 0.08 mM cellobiose), and pH (6.0). Using optimized assay an error prone PCR gene library of tmCDH was screened. Two mutants with 5 (H5) and 7 mutations (H9) were found having two times higher activity than the parent tmCDH enzyme that already had improved activity compared to wild type CDH whose activity could not be detected on the surface of yeast cells.",
publisher = "MDPI, Basel",
journal = "Applied Sciences-Basel",
title = "Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay",
number = "7",
volume = "9",
doi = "10.3390/app9071413"
}

Blazic, M., Balaž, A. M., Prodanović, O., Popović, N., Ostafe, R., Fischer, R.,& Prodanović, R.. (2019). Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay. in Applied Sciences-Basel
MDPI, Basel., 9(7).
https://doi.org/10.3390/app9071413

Blazic M, Balaž AM, Prodanović O, Popović N, Ostafe R, Fischer R, Prodanović R. Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay. in Applied Sciences-Basel. 2019;9(7).
doi:10.3390/app9071413 .

Blazic, Marija, Balaž, Ana Marija, Prodanović, Olivera, Popović, Nikolina, Ostafe, Raluca, Fischer, Rainer, Prodanović, Radivoje, "Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay" in Applied Sciences-Basel, 9, no. 7 (2019),
https://doi.org/10.3390/app9071413 . .