Uv 2600 uv vis spectrophotometer

A three-plasmid co-expression system was used to test the lyase activities of MpeQ and MpeW. Specifically, MpeQ or MpeW was co-expressed in E. coli BL21(DE3) cells with MpeA (pCOLA-Duet vector) along with a pACYC-Duet vector carrying heme oxygenase (HO1) and PEB synthase (PebS) genes (Kronfel et al., 2019 ). His-tagged MpeQ or non-tagged MpeW was then co-purified with MpeA using Co2+-affinity column (Talen). Eluted fractions were then examined by SDS polyacrylamide electrophoresis (SDS-PAGE) and absorption spectroscopy, and/or fluorescence spectroscopy. The bilin lyase activity was measured by the chromophorylated MpeA detected either by Zn-fluorescence (Berkelman and Lagarias, 1986 (link)), absorption spectroscopy (Shimadzu UV-2600 UV-Vis spectrophotometer), and/or fluorescence spectroscopy (Perkin Elmer LS55 fluorescence spectrophotometer). MpeA-PUB and MpeA-PEB have characteristic absorption peaks at 495 nm and 550 nm, respectively, while their corresponding emission peaks are around 500 nm and 565 nm. The lyase activities of the wild type MpeQ and mutants were quantified and compared by the absorbance readings at 495 nm normalized by the corresponding MpeA band intensities detected by SDS-PAGE (ImageJ). The relative activity of a specific mutant (in percentage) was calculated in comparison to the wild type for which the lyase activity was set to 100%.