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Cloning and characterization of a cDNA for 1-aminocyclopropane-1-carboxylate oxidase from papaya fruit

Lin, C.T., Lin, M.T., Shaw, J.F.
Journal of agricultural and food chemistry 1997 v.45 no.2 pp. 526-530
1-aminocyclopropane-1-carboxylic acid, Carica papaya, ripening, ethylene production, oxidoreductases, complementary DNA, nucleotide sequences, amino acid sequences, clones, recombinant DNA, genetic transformation, gene expression, Escherichia coli, sequence alignment, aminocyclopropanecarboxylate oxidase
A full-length complementary DNA (cDNA) clone encoding a putative 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase) from papaya was amplified by polymerase chain reaction technique from cDNAs synthesized from messenger RNA. Nucleotide sequence analysis of this cDNA clone revealed that it comprised a complete open reading frame coding for 310 amino acid residues. The deduced amino acid sequence showed high identity (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, H-39, H-177, D-179, L-195, Q-196, G-218, H-234, R-244, and S-246) are conserved as they are among all enzymes that require ferrous ion and ascorbate for activity. These suggest that the papaya cDNA clone encodes a cytosolic ACC oxidase. Furthermore, the coding region of ACC oxidase cDNA from papaya was introduced into an expression vector, pET-20b(+), and transformed into Escherichia coli BL21(DE3). A 0.45 mL enzyme crude extract from 5 mL culture in a typical assay produced 42 ppm of ethylene. A 38 kDa ACC oxidase protein was detected as the distinctive protein by Coomassie blue staining of SDS-PAGE, and western blot immunoanalysis confirmed the results of Coomassie blue staining. These indicate that this ACC oxidase cDNA clone can express active ACC oxidase enzyme in the E. coli system.