Uv 2070 plus detector

Recombinant CWPO-C (rCWPO-C) was prepared and purified according to the described methods by Shigeto et al. (2012 (link)). The oxidation of IAA by rCWPO-C was carried out in a 100 mM sodium acetate buffer (pH 5) containing 500 µM H₂O₂, 500 µM IAA, 2 U/ml rCWPO-C and rotary-incubated (Rotaflex, Argos) at 10 rpm for 0, 60, 120, 360 min at 25 °C. One U of CWPO-C oxidation activity was defined as the formation of the oxidation product from 2,6-dimethoxyphenol (2,6-DMP) in one µmol/min (Shigeto et al. 2012 (link)). As control, the reaction was carried out in the absence of rCWPO-C. Reaction mix was filtered with a Minisart SRP 4 PTFE-membrane and an aliquot of reaction mixture (20 µl) was analyzed by reverse-phase HPLC on a InertSustain AQ-C₁₈ column (100 nm, 5 μm, 4.6 × 150 mm; GL Sciences, Japan) using an isocratic elution buffer containing methanol : 1% formic acid mixture (40:60, v/v) at a flow rate of 1.0 ml/min. The eluted products were monitored at the absorbance of 250 nm using a Jasco UV-2070 Plus detector.