title: Expression, purification, and characterization of cold-adapted inorganic pyrophosphatase from psychrophilic Shewanella sp. AS-11.
abstract: In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn²⁺ over Mg²⁺ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.
authors.affiliation: a Department of Applied Biochemistry and Food Science , Saga University , Saga , Japan.
authors.forename: Elvy Like
authors.initials: EL
authors.lastname: Ginting
authors.forename: Syouhei
authors.initials: S
authors.lastname: Iwasaki
authors.forename: Chihiro
authors.initials: C
authors.lastname: Maeganeku
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Motoshima
authors.forename: Keiichi
authors.initials: K
authors.lastname: Watanabe
mesh_terms: D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003080:Cold Temperature; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology; D043564:Inorganic Pyrophosphatase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020592:Shewanella / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; D008345:Manganese; D043564:Inorganic Pyrophosphatase; D008274:Magnesium