Title |
Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae |
Author |
Vanessa Sayuri Sato, Renato F. Galdiano Júnior, Gisele Regina Rodrigues, Eliana G. M. Lemos, and João Martins Pizauro Junior* |
Address |
Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Tecnologia, 14884 900, Jaboticabal, São Paulo, Brasil |
Bibliography |
Journal of Microbiology, 54(2),106-113, 2016,
|
DOI |
10.1007/s12275-015-5354-3
|
Key Words |
acid ectophosphatase, Enterobacter asburiae, inhibition,
p-nitrophenylphosphate, ATPase, pyrophosphatase |
Abstract |
Expression of acid ectophosphatase by Enterobacter asburiae,
isolated from Cattleya walkeriana (Orchidaceae) roots
and identified by the 16S rRNA gene sequencing analysis,
was strictly regulated by phosphorus ions, with its optimal
activity being observed at an inorganic phosphate concentration
of 7 mM. At the optimum pH 3.5, intact cells released
p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate
(pNP)/min/108 cells. The membrane-bound enzyme
was obtained by centrifugation at 100,000 × g for 1 h
at 4°C. p-Nitrophenylphosphate (pNPP) hydrolysis by the
enzyme follows “Michaelis-Menten” kinetics with V = 61.2
U/mg and K0.5 = 60 μM, while ATP hydrolysis showed V =
19.7 U/mg, K0.5 = 110 μM, and nH = 1.6 and pyrophosphate
hydrolysis showed V = 29.7 U/mg, K0.5 = 84 μM, and nH =
2.3. Arsenate and phosphate were competitive inhibitors
with Ki = 0.6 mM and Ki = 1.8 mM, respectively. p-Nitrophenyl
phosphatase (pNPPase) activity was inhibited by
vanadate, while p-hydroxymercuribenzoate, EDTA, calcium,
copper, and cobalt had no inhibitory effects. Magnesium ions
were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production
of an acid ectophosphatase can be a mechanism for the solubilization
of mineral phosphates by microorganisms such
as Enterobacter asburiae that are versatile in the solubilization
of insoluble minerals, which, in turn, increases the availability
of nutrients for plants, particularly in soils that are
poor in phosphorus. |