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

Authors: Marija Blažić; Ana Marija Balaž; Olivera Prodanović; Nikolina Popović; Raluca Ostafe; Rainer Fischer; Radivoje Prodanovic;

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

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.

Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/2912]

Countries
Germany, Serbia
Related Organizations
Subjects by Vocabulary

Microsoft Academic Graph classification: Cellobiose chemistry.chemical_compound chemistry.chemical_classification biology Chemistry Directed evolution Biochemistry Cellobiose dehydrogenase Resazurin Enzyme assay Yeast Lactobionic acid Enzyme biology.protein

Library of Congress Subject Headings: lcsh:Technology lcsh:Chemistry lcsh:QH301-705.5 lcsh:QC1-999 lcsh:T lcsh:Biology (General) lcsh:QD1-999 lcsh:TA1-2040 lcsh:Engineering (General). Civil engineering (General) lcsh:Physics

Dewey Decimal Classification: ddc:600

Keywords

Technology, yeast surface display, resazurin, General Materials Science, Biology (General), cellobiose dehydrogenase, Instrumentation, Fluid Flow and Transfer Processes, T, Physics, General Engineering, 600, Engineering (General). Civil engineering (General), Computer Science Applications, Chemistry, info:eu-repo/classification/ddc/600, TA1-2040, QH301-705.5, QC1-999, QD1-999, flow cytometry, Process Chemistry and Technology, fluorescent assay

39 references, page 1 of 4

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This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).
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This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.
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