Biochemical characterization and expression of recombinant ACC oxidase in Escherichia coli and endogenous ACC oxidase from kiwifruit

To use recombinant 1-aminocyclopropane-1-carboxylate (ACC) oxidase for research on ethylene biosynthesis, the biochemistry of both recombinant and endogenous ACC oxidase from kiwifruit was compared. When induced by the addition of isopropyl-beta-D-thiogalactopyranoside (IPTG) to Escherichia coli (E. coli) cells transformed with cDNA AD-ACO1 using the pGEX-4T-1 vector, ACC oxidase identical to that from kiwifruit was expressed as a polypeptide of 37 kDa. Apparent K-m values for ACC for both recombinant and endogenous ACC oxidase were 41 and 16 mu M, respectively. Both forms of ACC oxidase exhibited absolute requirements for ferrous iron, ascorbate and bicarbonate for maximum activity The activities of both enzymes were inhibited by ethylenediaminetetraacetic acid (EDTA), 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron), o-phenanthroline (PA), alpha-aminoisobutyric acid (AIB), and p-chloromercuriphenylsulfonic acid (PCMPS). Addition of dithiothreitol (DTT) stimulated activity of both ACC oxidases. The results indicate that the recombinant ACC oxidase was similar biochemically to the endogenous kiwifruit enzyme. Western blot analysis using antibody raised against purified transformed ACC oxidase protein showed differential expression of endogenous ACC oxidase protein in kiwifruit during ripening. Expression may start in the columella region and then increase in surrounding tissues with progressive stages of ripening. (C) 1998 Elsevier Science B.V. All rights reserved.