Ultra-high-throughput screening method for the directed evolution of glucose oxidase.


Glucose oxidase (GOx) is used in many industrial processes that could benefit from improved versions of the enzyme. Some improvements like higher activity under physiological conditions and thermal stability could be useful for GOx applications in biosensors and biofuel cells. Directed evolution is one of the currently available methods to engineer improved GOx variants. Here, we describe an ultra-high-throughput screening system for sorting the best enzyme variants generated by directed evolution that incorporates several methodological refinements: flow cytometry, in vitro compartmentalization, yeast surface display, fluorescent labeling of the expressed enzyme, delivery of glucose substrate to the reaction mixture through the oil phase, and covalent labeling of the cells with fluorescein-tyramide. The method enables quantitative screening of gene libraries to identify clones with improved activity and it also allows cells to be selected based not only on the overall activity but also on the specific activity of the enzyme.

Submission Details

ID: Hiy8xcAG4

Submitter: Shu-Ching Ou

Submission Date: Dec. 4, 2018, 9:41 a.m.

Version: 1

Publication Details
Ostafe R;Prodanovic R;Nazor J;Fischer R,Chem Biol (2014) Ultra-high-throughput screening method for the directed evolution of glucose oxidase. PMID:24613019
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Glucose oxidase P13006 GOX_ASPNG