Purification and biochemical characterization of thermostable alkaline phosphatases produced by Rhizopus microsporus var. rhizopodiformis

• Published in: Folia microbiologica 2008-01, Vol.53 (6), p.509-516
• Main Authors: Barbosa Junior, A.,Universidade de Sao Paulo (Brazil). Departamento De Bioquimica e Imunologia, Guimaraes, L.H.S.,Universidade de Sao Paulo (Brazil). Departamento De Biologia, Terenzi, H.F.,Universidade de Sao Paulo (Brazil). Departamento De Biologia, Jorge, J.A.,Universidade de Sao Paulo (Brazil). Departamento De Biologia, Leone, F.A.,Universidade de Sao Paulo (Brazil). Departamento De Quimica, Polizeli, M.L.T.M.,Universidade de Sao Paulo (Brazil). Departamento De Biologia
• Format: Article
• Language: English
• Subjects: ACTIVADOR ENZIMATICO; ACTIVATEUR D'ENZYME; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALKALINE PHOSPHATASE; Alkaline Phosphatase - isolation & purification; Alkaline Phosphatase - metabolism; ALUMINIO; ALUMINIUM; Applied Microbiology; BIOCHEMISTRY; BIOCHIMIE; Biomedical and Life Sciences; BIOQUIMICA; Biotechnology; Cations - pharmacology; Cellulose; Chromatography, Agarose; Chromatography, DEAE-Cellulose; Chromatography, Gel; CINC; CULTURE MEDIA; ELECTROFORESIS; ELECTROPHORESE; ELECTROPHORESIS; Electrophoresis, Polyacrylamide Gel; Environmental Engineering/Biotechnology; ENZYME ACTIVATORS; ENZYME ACTIVITY; ESTABILIDAD; FOSFATASA ALCALINA; Fungal Proteins - isolation & purification; Fungal Proteins - metabolism; HIDROLISIS; Hot Temperature; http://www.fao.org/aos/agrovoc#c_10204; http://www.fao.org/aos/agrovoc#c_24940; http://www.fao.org/aos/agrovoc#c_2523; http://www.fao.org/aos/agrovoc#c_2604; http://www.fao.org/aos/agrovoc#c_267; http://www.fao.org/aos/agrovoc#c_27513; http://www.fao.org/aos/agrovoc#c_317; http://www.fao.org/aos/agrovoc#c_36930; http://www.fao.org/aos/agrovoc#c_4517; http://www.fao.org/aos/agrovoc#c_4570; http://www.fao.org/aos/agrovoc#c_4892; http://www.fao.org/aos/agrovoc#c_5753; http://www.fao.org/aos/agrovoc#c_6377; http://www.fao.org/aos/agrovoc#c_6568; http://www.fao.org/aos/agrovoc#c_7145; http://www.fao.org/aos/agrovoc#c_7657; http://www.fao.org/aos/agrovoc#c_8517; http://www.fao.org/aos/agrovoc#c_910; Hydrogen-Ion Concentration; HYDROLYSE; HYDROLYSIS; Immunology; Life Sciences; MAGNESIO; MAGNESIUM; MANGANESE; MANGANESO; MEDIO DE CULTIVO; Microbiology; MILIEU DE CULTURE; MOLECULAR WEIGHT; PESO MOLECULAR; PHOSPHATASE ALCALINE; POIDS MOLECULAIRE; PURIFICACION; PURIFICATION; RHIZOPUS; Rhizopus - enzymology; Rhizopus microsporus; SODIO; SODIUM; STABILITE; STABILITY; TEMPERATURA; TEMPERATURE; Triticum aestivum; Wheat bran; ZINC
• ISSN: 0015-5632; 1874-9356
• DOI: 10.1007/s12223-008-0080-4

The purification and kinetic characterization of intra- and extracellular alkaline phosphatases (AIPs) produced by the thermotolerant fungus Rhizopus microsporus var. rhizopodiformis is described in this article. High enzymic levels were produced within 1-2 d in agitated cultures with 1% of wheat bran. Intra- and extracellular AIPs 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. Mn2+, Na+ and Mg2+ stimulated the activity, while Al3+ and Zn2+ activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 deg C and pH 8.0, resp. The enzymes were stable at 50 deg C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a Km 0.28 and 0.22 mmol/L, with Vlim 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AIPs may be suitable for biotechnological applications.