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

Nikolina Popović,Raluca Ostafe,Rainer Fischer,Olivera Prodanović,Marija Blažić,Ana Balaž,Radivoje Prodanović

doi:10.3390/app9071413

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 (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.

Highlights

The cellobiose dehydrogenase (CDH; EC 1.1.99.18) is a monomeric, an extracellular hemoflavoprotein with two domains, flavin and heme domain, connected via flexible peptide linker [1].CDH catalyzes the oxidation of the reducing end of cellobiose and other β-1,4-linked disaccharidesAppl
Resazurin was used previously as a probe for assessing cell viability during which it was reduced to fluorescent resorufin [24] and in this article it was tried as an electron acceptor in CDH-catalyzed oxidation of lactose, Scheme 1
The assay was optimized for detection of CDH expressed on the surface of yeast cells

Summary

Introduction

The cellobiose dehydrogenase (CDH; EC 1.1.99.18) is a monomeric, an extracellular hemoflavoprotein with two domains, flavin and heme domain, connected via flexible peptide linker [1].CDH catalyzes the oxidation of the reducing end of cellobiose and other β-1,4-linked disaccharidesAppl. CDH catalyzes the oxidation of the reducing end of cellobiose and other β-1,4-linked disaccharides. Sci. 2019, 9, 1413 with a β-glucose moiety, to their corresponding lactones [2]. Different electron acceptors can reoxidize the reduced enzyme, such as 2,6-dicholoro-indophenol (DCIP), or metal ions, for example Fe(III). It has been suggested that CDH plays a role in lignocellulosic biomass hydrolysis, such as relief of product inhibition of cellulases by oxidation of cellobiose, generation of peroxide for inhibition of opposing microbes, and regeneration of redox metal ions (such as Fe2+ ) to sustain the Fenton-type chemistry that produces hydroxyl radicals who can modify polysaccharides causing partial hydrolysis and swelling of insoluble fibers which helps breakdown of lignocellulose by glycoside hydrolase [1,2,3]. CDH has been applied in biosensors for detection of lactose, in enzymatic biofuel cells as anode catalyst [4,5], for lactobionic acid production [6], dye removal [7], and bioremediation [1]

Methods

Results

Discussion

Conclusion