CLONING AND CHARACTERIZATION OF A CDNA FOR 1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE FROM PAPAYA FRUIT

A full-length complementary DNA (cDNA) clone encoding a putative 1-ami nocyclopropane-1-carboxylate oxidase (ACC oxidase) from papaya was amp lified by polymerase chain reaction technique from cDNAs synthesized f rom messenger RNA. Nucleotide sequence analysis of this cDNA clone rev ealed that it comprised a complete open reading frame coding for 310 a mino acid residues. The deduced amino acid sequence showed high identi ty (72-80%) with the sequence of ACC oxidase from other plant species. No transit peptide was found. The 12 residues (P-5, A-27, G-32, 11-39 , H-177, D-179, L-195, Q-196, G-218, H-234, R-244, and S-246) are cons erved as they are among all enzymes that require ferrous ion and ascor bate for activity. These suggest that the papaya cDNA clone encodes a cytosolic ACC oxidase. Furthermore, the coding region of ACC oxidase c DNA from papaya was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). A 0.45 mt enzyme crud e extract from 5 mt culture in a typical assay produced 42 ppm of ethy lene. A 38 kDa ACC oxidase protein was detected as the distinctive pro tein by Coomassie blue staining of SDS-PAGE, and western blot immunoan alysis confirmed the results of Coomassie blue staining. These indicat e that this ACC oxidase cDNA clone can express active ACC oxidase enzy me in the E. coli system.