Characterization of Mg-dechelatase activity obtained from Fragaria x ananassa fruit

Magnesium-dechelatase was obtained from strawberries (Fragaria x ananassa) by extraction with non-ionic detergents and concentrated by ammonium sulfate fractionation. Extracts with Mg-dechelatase activity were dissolved in high or low ionic strength and their heat stability and capacity to filtrate through 10-kDa cut-off membranes were analyzed. At high ionic strength, the extract was inactivated after 10 min at 100degreesC and no activity was found in the filtrate. On the contrary, at low ionic strength, the extract was not inactivated by the heat treatment and the filtrate showed Mg-dechelatase activity. Therefore, two kinds of extract were prepared and characterized: extract in high ionic strength (HMDS) and extract in low ionic strength filtered through 10-kDa cut-off (LMDS). The highest Mg-dechelatase activity of HMDS extract was found at 50 C, while the activity of LMDS extract increased continuously with temperature. Both extracts showed an optimum pH equal to 7.1 in phosphate buffer and 8.6 in Tris-tricine buffer. The apparent Km values, using chlorophyllin as the substrate, were 81 and 116 nM for HMDS and LMDS, respectively, indicating that HMDS showed a higher affinity toward the substrate. The Mg-dechelatase activity was enhanced by EDTA, reduced by Ca2+ and Mg2+, and strongly inhibited by Hg2+. Natural phenolic compounds either enhanced or reduced the activity. The analysis by molecular exclusion chromatography of a partially purified extract indicated that the Mg-dechelatase activity was associated to a compound with a molecular mass of 2180 +/- 20 Da. The treatment of partially purified extracts with proteinase K reduced significantly the activity. During strawberry fruit ripening, the Mg-dechelatase activity decreased from `small green' to `large green' stage and then increased again in `mature' fruit. (C) 2002 Editions scientifiques et medicales Elsevier SAS. All rights reserved.