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.
Submitter: Shu-Ching Ou
Submission Date: Dec. 4, 2018, 9:41 a.m.
|Number of data points||84|
|Assays/Quantities/Protocols||Experimental Assay: Thermal Stability: t1/2 ; Experimental Assay: Kinetic Constant: kcat: PBS ; Experimental Assay: Kinetic Constant: Km: PBS ; Experimental Assay: Kinetic Constant: kcat: Acetate ; Experimental Assay: Kinetic Constant: Km: Acetate ; Derived Quantity: SD of Kinetic Constant: Km: Acetate ; Derived Quantity: SD of Kinetic Constant: kcat: Acetate ; Derived Quantity: Kinetic Constant: kcat/Km (Specificity): Acetate ; Derived Quantity: SD of Kinetic Constant: Km: PBS ; Derived Quantity: Kinetic Constant: kcat/Km (Specificity): PBS ; Derived Quantity: SD of Thermal Stability: t1/2 ; Derived Quantity: SD of Kinetic Constant: kcat: PBS|
|Libraries||Variants for 1GAL_ABTS ; Variants for 1GAL|
|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|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|