title: Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae.
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/10(8) 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 K i = 0.6 mM and K i = 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.
authors.affiliation: Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Tecnologia, 14884 900, Jaboticabal, São Paulo, Brasil.
authors.forename: Vanessa Sayuri
authors.initials: VS
authors.lastname: Sato
authors.affiliation: Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Tecnologia, 14884 900, Jaboticabal, São Paulo, Brasil.
authors.forename: Renato F
authors.initials: RF
authors.lastname: Galdiano Júnior
authors.affiliation: Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Tecnologia, 14884 900, Jaboticabal, São Paulo, Brasil.
authors.forename: Gisele Regina
authors.initials: GR
authors.lastname: Rodrigues
authors.affiliation: Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Tecnologia, 14884 900, Jaboticabal, São Paulo, Brasil.
authors.forename: Eliana G M
authors.initials: EG
authors.lastname: Lemos
authors.affiliation: Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal, Departamento de Tecnologia, 14884 900, Jaboticabal, São Paulo, Brasil. jpizauro@fcav.unesp.br.
authors.forename: João Martins
authors.initials: JM
authors.lastname: Pizauro Junior
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D016000:Cluster Analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004754:Enterobacter / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D029595:Orchidaceae / Q000382:microbiology; D009943:Organophosphorus Compounds / Q000378:metabolism; D010758:Phosphorus / Q000378:metabolism*; D010802:Phylogeny; D018517:Plant Roots / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D004791:Enzyme Inhibitors; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D012336:RNA, Ribosomal, 16S; D010758:Phosphorus; C008644:nitrophenylphosphate; D000255:Adenosine Triphosphate; D000135:Acid Phosphatase
keywords: ATPase; Enterobacter asburiae; acid ectophosphatase; inhibition; p-nitrophenylphosphate; pyrophosphatase
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