Fig. 4From: A novel hydroxycinnamoyl transferase for synthesis of hydroxycinnamoyl spermine conjugates in plantsSubstrate specificity and pH optima of SrSpmHT. a, Isolation of recombinant S. richardii spermine hydroxycinnamoyl tansferase (SrSpmHT). His-tagged recombinant proteins were expressed in E. coli and purified from cell lysates by nickel affinity chromatography. Evaluation of protein purity was performed by SDS-PAGE with coomassie blue staining of the gel (left), and Western blotting using anti-His-tag antibody (right): T, total cell lysate; P, cell pellet; S, supernatant; F, Flowthrough solution; W, last column wash; E, column eluates; b, Acyl donor substrate specificity of SrSpmHT. The activities were measured at 120 μM hydroxycinnaminoyl CoA (caffeoyl CoA, feruloyl-CoA and ρ-coumaroyl CoA) and 2.5 mM polyamine (spermidine, Spd; spermine, Spm; putrescine, Put, octopamine, Oct; tyramine, Tyr); c, C18-HPLC-DAD chromatogram of reaction products of SrSpmHT with spermine plus ρ-coumaroyl CoA as substrates (mAU, milli-absorption units). N1-ρ-coumaroyl-Spm was used as a standard; d, UV absorbance spectra of ρ-coumaroyl CoA (substrate) and N1-ρ-coumaroyl-Spm (product); e, pH-dependent activities of SrSpmHT. Reactions were performed with 60 μM caffeoyl CoA and 2.5 mM spermine at different pH environments. Bars show standard errors calculated from triplicatesBack to article page