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


Abstract

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 PDB Entries

Structure ID Release Date Resolution Structure Title
3QVP 2011-06-01 1.2 Crystal structure of glucose oxidase for space group C2221 at 1.2 A resolution
3QVR 2011-06-01 1.3 Crystal structure of glucose oxidase for space group P3121 at 1.3 A resolution.
5NIW 2017-11-15 1.8 Glucose oxydase mutant A2
5NIT 2017-11-15 1.87 Glucose oxidase mutant A2
1CF3 1999-03-26 1.9 GLUCOSE OXIDASE FROM APERGILLUS NIGER
1GAL 1993-10-31 2.3 CRYSTAL STRUCTURE OF GLUCOSE OXIDASE FROM ASPERGILLUS NIGER: REFINED AT 2.3 ANGSTROMS RESOLUTION

Relevant UniProtKB Entries

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