Figure 5.

In Vitro Chlorophyll Dephytylase Activity of Recombinant CLD1 Expressed in E. coli Cells.

(A) HPLC trace of chlorophyll a (Chl a), chlorophyll b (Chl b), or Phein a after being incubated with the recombinant wild-type or mutated CLD1 proteins (G193D and S165A) in reaction mixtures contained 55 μM of each substrate and 5% acetone. The reactions were conducted at 35°C for 60 min.

(B) The effect of substrate concentration on the initial velocity of the wild type (upper panel) and G193D enzyme (lower panel). The substrate of indicated concentration was incubated with 0.4 or 0.08 μg of wild-type or G193D protein, respectively, at 35°C for 30 min. The reactions were stopped by adding acetone to the reaction mixtures, and the dephytylated products were quantified by HPLC. The velocity (v) is expressed as nmol of dephytylated product formed per min per mg CLD1 protein. Data are means ± sd from three replicates.