Vanadium bromoperoxidase-coupled fluorescent assay for flow cytometry sorting of glucose oxidase gene libraries in double emulsions.
A Vanadium bromoPeroxidase-coupled fluorescent assay (ViPer) for ultrahigh-throughput screening of glucose oxidase (GOx) gene libraries employing double emulsions and flow cytometry was developed. The assay is based on detection of the product of a GOx reaction, hydrogen peroxide, that is first converted to a hypobromide by vanadium bromoperoxidase in the presence of sodium bromide. The hypobromide is afterwards detected in a reaction with a fluorogenic probe, 3-carboxy-7-(4'-aminophenoxy)-coumarine, where fluorescent 3-carboxy-coumarine is released. The ViPer screening system is three times more sensitive than a horseradish peroxidase coupled detection system and more resistant to bleaching of fluorescence in excess of peroxide. Using the ViPer screening system a high epPCR gene library containing 100,000 different GOx variants was screened for active clones in less than 1 h by flow cytometry. A library containing 0.15% of yeast cells expressing active enzyme variants and with an average GOx activity in the liquid culture of 0.47 U/mL, after one round of sorting, had 28.12% of the yeast cells expressing the active GOx (an enrichment factor of 200) and 26.8 U/mL of the GOx activity in the liquid culture (an enrichment factor of 57). The developed screening system could be adapted and used in a directed evolution of GOx and other hydrogen peroxide-producing enzymes (oxidases) and glycosidases if coupled with a carbohydrate oxidase.
Jacobs University Bremen, Bremen, Germany. rprodano@chem.bg.ac.rs
Radivoje
R
Prodanovic
Raluca
R
Ostafe
Milan
M
Blanusa
Ulrich
U
Schwaneberg
D004655:Emulsions / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D015870:Gene Expression; D015723:Gene Library; D005949:Glucose Oxidase / Q000235:genetics* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000032:analysis / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004655:Emulsions; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D010544:Peroxidases; C026195:bromide peroxidase