PURIFICATION AND BIOCHEMICAL-COMPARISON OF 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID DEAMINASE PROTEINS EXPRESSED IN DELAYED RIPENING TOMATO AND ESCHERICHIA-COLI - STUDIES FOR A FOOD SAFETY ASSESSMENT

Tomato plants with delayed fruit ripening have been developed by intro duction of the gene encoding the 1-aminocyclopropane-1-carboxylic acid deaminase (ACCd) protein into the tomato genome. The ACCd enzyme dela ys fruit ripening by decreasing ethylene synthesis. To facilitate safe ty assessments of the ACCd protein, the identical coding sequence used for tomato transformation was introduced into Escherichia coli to pro duce gram quantities of the protein. A method that included hydrophobi c interaction and anion exchange chromatography was developed to purif y approximately 2.5 g of ACCd. The E. coli-produced ACCd protein was d emonstrated to be chemically and functionally equivalent to the ACCd p rotein expressed in and purified from delayed ripening tomato fruit. T he proteins from each source had comparable apparent molecular weights , immunoreactivities, primary amino acid sequences, and enzymatic acti vities, and both proteins lacked glycosylation. These data validate th e use of E. coli-produced ACCd protein to assess the safety of ACCd pr oduced in delayed ripening tomatoes.