title: Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings.
abstract: Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.
authors.affiliation: Department of Biological Science, Silla University, Busan 617-736, Korea.
authors.forename: Donggiun
authors.initials: D
authors.lastname: Kim
authors.forename: Gunsup
authors.initials: G
authors.lastname: Lee
authors.forename: Man
authors.initials: M
authors.lastname: Chang
authors.forename: Jongbum
authors.initials: J
authors.lastname: Park
authors.forename: Youngjae
authors.initials: Y
authors.lastname: Chung
authors.forename: Sukchan
authors.initials: S
authors.lastname: Lee
authors.forename: Taek-Kyun
authors.initials: TK
authors.lastname: Lee
mesh_terms: D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D018532:Pisum sativum / Q000201:enzymology*; D011887:Raffinose / Q000378:metabolism; D036226:Seedlings / Q000201:enzymology*; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D013395:Sucrose; D043324:beta-Fructofuranosidase; D011887:Raffinose