Activities of muteins were additionally confirmed with HPLC analysis. The reaction mixture consisted of 0.1 M KH2PO4/K2HPO4 buffer pH 6.5, 400 μM DCPIP, 200 μM AD in IPA (2%) and 24.6 -38.9 μM of AcmB. The reactions were carried out in a thermoblock at 30° C and 800 rpm for 30 minutes. The reaction progress was stopped after 30 minutes by mixing the samples with acetonitrile (1:1), followed by centrifugation at 14 000 g for 5 minutes and analysis with LC DAD-HPLC (Agilent 1100) according to Wojtkiewicz et al. .