To determine protein concentrations at different steps of enzyme purification, the Bradford method was used employing bovine serum albumin as standard protein (Bradford 1976 (link)). The measurements were carried out at room temperature in 96-well micro titer plates using an Infinite M200 Pro plate reader (Tecan, Männedorf, Switzerland). The molar extinction coefficients of purified PeAAO1 variants were calculated after heat denaturation of the samples and detection of released FAD as described for PeAAO2 wild-type (Jankowski et al. 2020 (link)) and used for determination of molar enzyme concentrations. Latter ones were used to calculate enzyme concentration in fermentation supernatant, specific activities of purified enzymes, and kinetic constants.
N-Deglycosylation was performed using 20 µg of purified enzymes and peptide-N-amidase PNGase F (New England Biolabs, Frankfurt am Main, Germany) under denaturing conditions. For this, the samples were boiled at 100 °C for 10 min in the presence of SDS prior to deglycosylation with PNGase F. An aliquot of the deglycosylated samples as well as of the purified enzymes (each 5 µg) were loaded onto a 12.5% resolving gel and SDS-PAGE was conducted according to the protocol of Laemmli (1970 (link)).
N-Deglycosylation was performed using 20 µg of purified enzymes and peptide-N-amidase PNGase F (New England Biolabs, Frankfurt am Main, Germany) under denaturing conditions. For this, the samples were boiled at 100 °C for 10 min in the presence of SDS prior to deglycosylation with PNGase F. An aliquot of the deglycosylated samples as well as of the purified enzymes (each 5 µg) were loaded onto a 12.5% resolving gel and SDS-PAGE was conducted according to the protocol of Laemmli (1970 (link)).
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