Convenient microtiter plate‐based, oxygen‐independent activity assays for flavin‐dependent oxidoreductases based on different redox dyes

Abstract

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs.

Engineering of pyranose 2-oxidase for modified oxygen reactivity

“…Cultivation, expression and sample preparation of the libraries were carried out according to Spadiut et al [47] without freezing step after cell lysis. The supernatant was used for the activity assay with peroxidase/ABTS and DCPIP as described in [47] and [48] with modifications: 20 mL of supernatant were mixed with 80 mL ABTS reaction mix (0.035 mg mL 21 horseradish peroxidase, 0.7 mg mL 21 in 50 mM phosphate buffer pH 6.5 and 100 mM Dglucose), or with 80 mL DCPIP reagent containing 0.054 mg mL 21 DCPIP in 50 mM phosphate buffer pH 6.5 and 100 mM D-glucose. The formation of ABTS +N was followed at 420 nm and DCPIP reduction was detected at 620 nm with a Sunrise TM microplate reader (Tecan, Mä nnedorf, CH).…”

Enzymes Suitable for Biorefinery to Coproduce Hexaric Acids and Electricity from Hexuronic Acids Derived from Biomass

“…Measurements of PQQ‐GDH were conducted at 25 °C and measurements of DH PDH were conducted at 30 °C to conform with the conditions in previous reports ,. Enzyme activity was calculated by using an extinction coefficient of DCPIP of 520 nm (6.6 mL mol −1 cm −1 was used for pH values above 6.5 and 6.8 mL mol −1 cm −1 was used when the pH value was below 6.5) and an extinction coefficient of MG of 655 nm (46.6 mL mol −1 cm −1 ) . The reaction mixture for substrate specificity tests contained each enzyme, 0.1 m of each substrate, 0.1 m phosphates (pH 7.0 for PQQ‐GDH and pH 7.5 for DH PDH ), 1 m m PMS, and 0.1 m m DCPIP in a total volume of 200 μL.…”

Electrochemical characterization of the pyranose 2-oxidase variant N593C shows a complete loss of the oxidase function with full preservation of substrate (dehydrogenase) activity

“…15,16 After concentration and washing steps, both POx enzymes (wt-POx and POx-C) were stored at 8 °C. The activity assay, applied to wild-type POx (wt-POx) and the mutant N593C (POx-C) was previously described.…”