Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay
Authors
Blažić, Marija
Balaž, Ana Marija

Prodanović, Olivera

Popović, Nikolina

Ostafe, Raluca

Fischer, Rainer

Prodanović, Radivoje

Article (Published version)
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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 (10mMlactose and 0.08mMcellobiose), 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 foun...d 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.
Keywords:
cellobiose dehydrogenase / resazurin / fluorescent assay / flow cytometry / yeast surface displaySource:
Applied Sciences, 2019, 9, 7, 1413-Publisher:
- MDPI
Funding / projects:
DOI: 10.3390/app9071413
ISSN: 2076-3417
WoS: 000466547500148
Scopus: 2-s2.0-85064083412
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IHTMTY - JOUR AU - Blažić, Marija AU - Balaž, Ana Marija AU - Prodanović, Olivera AU - Popović, Nikolina AU - Ostafe, Raluca AU - Fischer, Rainer AU - Prodanović, Radivoje PY - 2019 UR - https://cer.ihtm.bg.ac.rs/handle/123456789/2875 AB - 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 (10mMlactose and 0.08mMcellobiose), 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 T2 - Applied Sciences T1 - Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay VL - 9 IS - 7 SP - 1413 DO - 10.3390/app9071413 ER -
@article{ author = "Blažić, Marija and Balaž, Ana Marija and Prodanović, Olivera and Popović, Nikolina and Ostafe, Raluca and Fischer, Rainer and Prodanović, Radivoje", year = "2019", abstract = "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 (10mMlactose and 0.08mMcellobiose), 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", journal = "Applied Sciences", title = "Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay", volume = "9", number = "7", pages = "1413", doi = "10.3390/app9071413" }
Blažić, 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 MDPI., 9(7), 1413. https://doi.org/10.3390/app9071413
Blažić 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. 2019;9(7):1413. doi:10.3390/app9071413 .
Blažić, 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, 9, no. 7 (2019):1413, https://doi.org/10.3390/app9071413 . .