Fig. 6 in Genome-wide identification and characterization of genes involved in melatonin biosynthesis in Morus notabilis (wild mulberry)

Fig. 6. Purification and enzymatic analyses of recombinant His-tagged MnCOMT1. A: Purification of recombinant His-tagged MnCOMT1 (lane1: Protein Marker; lane2: pCold TF (+IPTG); lane3: recombinant protein (-IPTG); lane4: recombinant protein (+IPTG); lane5: purified protein). B: Mass spectrum of compounds detected using UPLC-MS/MS. a: melatonin standard; b: Chromatogram of N-acetylserotonin catalyzed by MnCOMT1. C: Determination of Km and Vmax of MnCOMT1 for Nacetylserotonin. MnCOMT1 (1.0 μg) was co-incubated with different concentrations of N-acetylserotonin for 20 min at 37 ◦C. D: Mass spectrum of compounds detected using UPLC-MS/MS. a: 5-methoxytryptamine standard; b: Chromatogram of serotonin catalyzed by MnCOMT1. E: Determination of Km and Vmax of MnCOMT1 for serotonin. MnCOMT1 (1.0 μg) was co-incubated with different concentrations of serotonin for 20 min at 37 ◦C. " IPTG" stand without addition of 1 mM IPTG to the E. coli BL21 (DE3) culture, "+IPTG" stand addition of 1 mM IPTG to the E. coli BL21 (DE3) culture. "TF" is Trigger Factor, a prokaryotic ribosomerelated molecular chaperone that facilitates the translation and folding of peptides. "*" indicates Trigger factor.