Biochemical characterization and expression of recombinant ACC oxidase in Escherichia coli and endogenous ACC oxidase from kiwifruit
Zhong Chuan Xu, Hiroshi Hyodo, Yoshinori Ikoma, Masamichi Yano and Kazunori Ogawa
Postharvest Biology and Technology. Volume 14, Issue 1, September 1998, Pages 41-50.
1998
บทคัดย่อ
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-
-
-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 Km values for
ACC for both recombinant and endogenous ACC oxidase were 41 and 16 
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),
-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.