Purification, Characterization, and Steady-State Kinetics of a meta-Cleavage Compound Hydrolase from Pseudomonas fluorescens IP01

概要

論文の詳細を見る
2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IPOl, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20℃. The enzyme has a K_m of 7.3 μM and a k_<cat> of 21 s^<-1> for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25℃), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodF. Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10^5-fold lower activity than that of the wild-type enzyme.
社団法人日本生物工学会の論文
2002-06-20