title: Purification and biochemical characterization of thermostable alkaline phosphatases produced by Rhizopus microsporus var. rhizopodiformis.
abstract: The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1-2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3x, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn(2+), Na(+) and Mg(2+) stimulated the activity, while Al(3+) and Zn(2+) activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 degrees C and pH 8.0, respectively. The enzymes were stable at 50 degrees C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K (m) 0.28 and 0.22 mmol/L, with upsilon (lim) 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade de São Paulo, Brazil.
authors.forename: A Barbosa
authors.initials: AB
authors.lastname: Junior
authors.forename: L H S
authors.initials: LH
authors.lastname: GuimarAes
authors.forename: H F
authors.initials: HF
authors.lastname: Terenzi
authors.forename: J A
authors.initials: JA
authors.lastname: Jorge
authors.forename: F A
authors.initials: FA
authors.lastname: Leone
authors.forename: M L T M
authors.initials: ML
authors.lastname: Polizeli
mesh_terms: D000469:Alkaline Phosphatase / Q000302:isolation & purification* / Q000378:metabolism; D002412:Cations / Q000494:pharmacology; D002847:Chromatography, Agarose; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012233:Rhizopus / Q000201:enzymology*
chemical_list: D002412:Cations; D005656:Fungal Proteins; D000469:Alkaline Phosphatase