CER - Central Repository
Institute of Chemistry, Technology and Metallurgy
    • English
    • Српски
    • Српски (Serbia)
  • English 
    • English
    • Serbian (Cyrilic)
    • Serbian (Latin)
  • Login
View Item 
  •   Central Repository
  • IHTM
  • Radovi istraživača / Researchers' publications
  • View Item
  •   Central Repository
  • IHTM
  • Radovi istraživača / Researchers' publications
  • View Item
JavaScript is disabled for your browser. Some features of this site may not work without it.

Directed Evolution of Cellobiose Dehydrogenase on the Surface of Yeast Cells Using Resazurin-Based Fluorescent Assay

Thumbnail
2019
osnovni rad (1.743Mb)
Authors
Blažić, Marija
Balaž, Ana Marija
Prodanović, Olivera
Popović, Nikolina
Ostafe, Raluca
Fischer, Rainer
Prodanović, Radivoje
Article (Published version)
Metadata
Show full item record
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 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 display
Source:
Applied Sciences, 2019, 9, 7, 1413-
Publisher:
  • Basel : MDPI
Projects:
  • Study of structure-function relationships in the plant cell wall and modifications of the wall structure by enzyme engineering (RS-173017)

DOI: 10.3390/app9071413

ISSN: 2076-3417

WoS: 000466547500148

Scopus: 2-s2.0-85064083412
[ Google Scholar ]
3
1
URI
http://cer.ihtm.bg.ac.rs/handle/123456789/2875
Collections
  • Radovi istraživača / Researchers' publications
Institution
IHTM

DSpace software copyright © 2002-2015  DuraSpace
About CeR – Central Repository | Send Feedback

OpenAIRERCUB
 

 

All of DSpaceInstitutionsAuthorsTitlesSubjectsThis institutionAuthorsTitlesSubjects

Statistics

View Usage Statistics

DSpace software copyright © 2002-2015  DuraSpace
About CeR – Central Repository | Send Feedback

OpenAIRERCUB