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

Author:
Lin, C.T., Lin, M.T., Shaw, J.F.
Source:
Journal of agricultural and food chemistry 1997 v.45 no.2 pp. 526-530
ISSN:
0021-8561
Subject:
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
Abstract:
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.
Agid:
1369314